Microbial consortia

ABSTRACT

Provided herein are microbial strains isolated de novo. In some instances, the bacterial strains include genera, species, and/or strains of Lactobacillus johnsonii, Lactobacillus crispatus, Faecalibacterium prausnitzii, Akkermansia muciniphila, Bifidobacterium longum, and/or Bifidobacterium longum infantis strains. These bacterial strains can be used in the treatment of dysbiosis, inflammation, and other disorders.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.17/067,354, filed Oct. 9, 2020, which is a continuation application ofPCT application serial number PCT/US2019/026674, filed Apr. 9, 2019,which claims the benefit of U.S. Provisional Application No. 62/655,562,filed Apr. 10, 2018, which are hereby incorporated by reference in theirentireties and for all purposes.

BACKGROUND

Recent developments in the areas of microbiome and genome researchprovide evidence that the microbial composition of the human gutfundamentally influences human health, disease onset and progression.However, much remains unknown with regards to the microbiome-hostrelationships, functional and metabolic changes in host due to themicrobiome composition, as well as the potential development ofmicrobial compositions for therapeutic applications.

SUMMARY

The present disclosure provides compositions, methods and kitscomprising one or more bacterial strains useful for the preventionand/or treatment of a disease or condition in a subject (e.g., a rodentor a human). In some cases, a composition comprising the at least onebacterial strain can be administered orally, and can be administered incombination with one or more pharmaceutically acceptable excipients. Insome cases, a therapeutic bacterial consortium (e.g., comprising anAkkermansia muciniphila strain, Faecalibacterium prausnitzii strain,Lactobacillus crispatus strain) of the present disclosure can beadministered to a subject in combination with one or more othertherapeutic agents (e.g., small molecule drug, therapeutic peptides orproteins, etc.).

In various aspects, the present disclosure provides a method of treatinginflammation or an inflammatory disease in a subject in need thereof,comprising administering to the subject an effective amount of abacterial population comprising one or more bacterial strains ofTable 1. In some cases, the one or more bacterial strains of Table 1comprise one or more bacterial strains associated with isolate numbers1-4. In some cases, the one or more bacterial strains of Table 1comprise one or more bacterial strains associated with isolate numbers5-40. In some cases, the one or more bacterial strains of Table 1comprise one or more bacterial strains associated with isolate numbers184-199. In some cases, the one or more bacterial strains of Table 1comprise one or more bacterial strains associated with isolate numbers214-216. In some cases, the one or more bacterial strains of Table 1comprise one or more bacterial strains associated with isolate numbers223-226. In some cases, the one or more bacterial strains of Table 1comprise one or more bacterial strains associated with isolate numbers111-115. In some cases, the inflammatory disease is an allergy, atopy,asthma, an autoimmune disease, an autoinflammatory disease, ahypersensitivity, pediatric allergic asthma, allergic asthma,inflammatory bowel disease, Celiac disease, Crohn's disease, colitis,ulcerative colitis, collagenous colitis, lymphocytic colitis,diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnantloop syndrome, chronic persistent diarrhea, intractable diarrhea ofinfancy, Traveler's diarrhea, immunoproliferative small intestinaldisease, chronic prostatitis, postenteritis syndrome, tropical sprue,Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis,Behcet's disease, uveitis, pyoderma gangrenosum, erythema nodosum,traumatic brain injury, psoriatic arthritis, juvenile idiopathicarthritis, multiple sclerosis, systemic lupus erythematosus (SLE),myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1,Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome,vasculitis, glomerulonephritis, auto-immune thyroiditis, bullouspemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison'sdisease, Vitiligo, acne vulgaris, pelvic inflammatory disease,reperfusion injury, sarcoidosis, transplant rejection, interstitialcystitis, atherosclerosis, or atopic dermatitis. In some cases, thesubject is less than about 24 months old. In some cases, the subject isa neonate.

In various aspects, the present disclosure provides a method of treatingdysbiosis in a subject in need thereof, comprising administering to thesubject an effective amount of a bacterial population comprising one ormore bacterial strains of Table 1. In some cases, the one or morebacterial strains of Table 1 comprise one or more bacterial strainsassociated with isolate numbers 1-4. In some cases, the one or morebacterial strains of Table 1 comprise one or more bacterial strainsassociated with isolate numbers 5-40. In some cases, the one or morebacterial strains of Table 1 comprise one or more bacterial strainsassociated with isolate numbers 184-199. In some cases, the one or morebacterial strains of Table 1 comprise one or more bacterial strainsassociated with isolate numbers 214-216. In some cases, the one or morebacterial strains of Table 1 comprise one or more bacterial strainsassociated with isolate numbers 223-226. In some cases, the one or morebacterial strains of Table 1 comprise one or more bacterial strainsassociated with isolate numbers 111-115. In some cases, the subject isless than about 24 months old. In some cases, the subject is a neonate.

In various aspects, the present disclosure provides s method of treatinga viral respiratory infection in a subject in need thereof, comprisingadministering to the subject an effective amount of a bacterialpopulation comprising one or more bacterial strains of Table 1. In somecases, the one or more bacterial strains of Table 1 comprise one or morebacterial strains associated with isolate numbers 1-4. In some cases,the one or more bacterial strains of Table 1 comprise one or morebacterial strains associated with isolate numbers 5-40. In some cases,the one or more bacterial strains of Table 1 comprise one or morebacterial strains associated with isolate numbers 184-199. In somecases, the one or more bacterial strains of Table 1 comprise one or morebacterial strains associated with isolate numbers 214-216. In somecases, the one or more bacterial strains of Table 1 comprise one or morebacterial strains associated with isolate numbers 223-226. In somecases, the one or more bacterial strains of Table 1 comprise one or morebacterial strains associated with isolate numbers 111-115. In somecases, the subject is less than about 24 months old. In some cases, thesubject is a neonate. In some cases, the bacterial population isformulated as an oral dosage form. In some cases, said oral dosage formfurther comprises an excipient. In some cases, said excipient comprisesan excipient that consumes oxygen. In some cases, said oral dosage formis in the form of a drop, a liquid, a frozen liquid, a suspension, anemulsion or a powder. In some cases, the bacterial population producesone or more biologically active compounds. In some cases, the one ormore biologically active compounds comprise one or more fatty acids, oneor more lipids, one or more phospholipids, a derivative of any of theabove, or any combination thereof In some cases, the one or more fattyacids comprise short-chain fatty acids. In some cases, the one or morephospholipids comprise one or more phosphatidylcholines, or one or morederivatives thereof.

In various aspects, the present disclosure provides a compositioncomprising one or more bacterial strains of Table 1. In some cases, suchcomposition can further comprise at least about 10⁷ cells of said one ormore bacterial strains of Table 1. In some cases, the composition canfurther comprise from about 10⁷ to about 10¹¹ cells, or from about 10⁸cells to about 10⁹ cells of said one or more bacterial strains ofTable 1. In some cases, the composition comprises three or morebacterial strains of Table 1. In some cases, said three or morebacterial strains of Table 1 act synergistically. In some cases, saidcomposition comprises four or more bacterial strains of Table 1. In somecases, said four or more bacterial strains of Table 1 actsynergistically. In some cases, said composition comprises five or morebacterial strains of Table 1. In some cases, said five or more bacterialstrains of Table 1 act synergistically. In some cases, said three ormore bacterial strains of Table 1 are substantially pure. In some cases,the composition comprises at least one strain of Lactobacillus johnsoniior Lactobacillus crispatus, at least one strain of Faecalibacteriumprausnitzii, and at least one strain of Akkermansia muciniphila, andoptionally at least one strain of a Bifidobacterium. In some cases, thecomposition comprises two or more distinct strains of Lactobacilluscrispatus. In some cases, the composition comprises two or more distinctstrains of Faecalibacterium prausnitzii. In some cases, the compositioncomprises two or more distinct strains of Akkermansia muciniphila. Insome cases, the composition comprises two or more distinct strains of aBifidobacterium.

In various aspects, the present disclosure provides a compositioncomprising three or more bacterial species of Table 1, wherein saidcomposition comprises three or more bacterial strains of Table 1.

In various aspects, the present disclosure provides a compositioncomprising four or more bacterial species of Table 1, wherein saidcomposition comprises four or more bacterial strains of Table 1.

In various aspects, the present disclosure provides a compositioncomprising one or more strains of Lactobacillus sp., Faecalibacteriumsp., or Akkermansia sp., which one or more strains are from Table 1. Insome cases, said composition comprises strains of two or more ofLactobacillus sp., Faecalibacterium sp., and Akkermansia sp., whichstrains are from Table 1. In some cases, said composition comprisesstrains of Lactobacillus sp., Faecalibacterium sp., and Akkermansia sp.,which strains are from Table 1. In some cases, said compositioncomprises any one of the strains Akkermansia muciniphila ST7,Faecalibacterium prausnitzii ST38, and Lactobacillus crispatus ST100, orany combination thereof. In some cases, said composition comprises thestrains Akkermansia muciniphila ST7, Faecalibacterium prausnitzii ST38,and Lactobacillus crispatus ST100. In some cases, the compositionproduces one or more biologically active compounds. In some cases, theone or more biologically active compounds comprise one or more fattyacids, one or more lipids, one or more phospholipids, a derivative ofany of the above, or any combination thereof. In some cases, the one ormore fatty acids comprise short-chain fatty acids. In some cases, theone or more phospholipids comprise one or more phosphatidylcholines, orone or more derivatives thereof. In some cases, said composition isformulated as an oral dosage form. In some cases, said oral dosage formfurther comprises an excipient. In some cases, said excipient comprisesan excipient that consumes oxygen. In some cases, said oral dosage formis in the form of a drop, a liquid, a frozen liquid, a suspension, anemulsion or a powder.

In various aspects, the present disclosure provides a containercomprising a composition comprising one or more bacterial strains ofTable 1. In some cases, such container comprises a compositioncomprising one or more of any one of the strains shown in Table 1. Insome cases, said composition is formulated in an aerosol, vapor, spray,or mist.

In various aspects, the present disclosure provides a kit comprising (1)a container comprising a composition comprising one or more bacterialstrains of Table 1, and (2) instructions directing a user to use saidcomposition. In some cases, the kit comprises a container as describedherein. In some cases, such container can be used for treatinginflammation or an inflammatory disease in a subject in need thereof.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A shows IgE concentration values (in ng/mL) in mouse plasmaobtained from three independent studies using the cockroach allergen(CRA) murine model. Non-treated (NT) mice, cockroach allergen (CRA)treated mice, and mice treated with CRA and an oral supplementationcontaining the microbial consortium (e.g., the therapeutic consortiumabbreviated as “TC3”) consisting of Lactobacillus johnsonii,Faecalibacterium prausnitzii, and Akkermansia muciniphila were used, inaccordance with the present disclosure.

FIG. 1B shows CRA-specific IgE concentration values (determined bymeasuring the optical density at 450 nm) in mouse plasma obtained fromthree independent studies in NT mice, CRA treated mice, and mice treatedwith CRA and an oral TC3 supplementation, in accordance with the presentdisclosure.

FIG. 1C shows histamine concentration values (in ng/mL) in mouse plasmaobtained from three independent studies in NT mice, CRA treated mice,and mice treated with CRA and an oral TC3 supplementation, in accordancewith the present disclosure.

FIG. 1D shows IL-4 concentration values (in pg/mL) in mouse lunghomogenate obtained from three independent studies in NT mice, CRAtreated mice, and mice treated with CRA and an oral TC3 supplementation,in accordance with the present disclosure.

FIG. 1E shows IL-13 concentration values (in pg/mL) in mouse lunghomogenate obtained from three independent studies in NT mice, CRAtreated mice, and mice treated with CRA and an oral TC3 supplementation,in accordance with the present disclosure.

FIG. 1F shows plasma concentration ratios of CRA-specific IgE toCRA-specific IgG3 obtained from three independent studies in NT mice,CRA treated mice, and mice treated with CRA and an oral TC3supplementation, in accordance with the present disclosure.

FIG. 1G shows the fold change of IL-4 concentration in lung tissuedetermined via qPCR and obtained from three independent studies in NTmice, CRA treated mice, and mice treated with CRA and an oral TC3supplementation, in accordance with the present disclosure.

FIG. 1H shows the fold change of IL-13 concentration in lung tissuedetermined via qPCR and obtained from three independent studies in NTmice, CRA treated mice, and mice treated with CRA and an oral TC3supplementation, in accordance with the present disclosure.

FIG. 1I shows the relative percentage of regulatory T (Tre_(reg)) cells(CD4⁺, CD127⁺, CD25⁺, Foxp3⁺ cells) in lung tissue obtained from threeindependent studies in NT mice, CRA treated mice, and mice treated withCRA and an oral TC3 supplementation, in accordance with the presentdisclosure.

FIG. 1J shows the relative percentage of type 2 T helper (T_(H)2) cells(CD3⁺, CD4⁺, ST2⁺ cells) in lung tissue obtained from three independentstudies in NT mice, CRA treated mice, and mice treated with CRA and anoral TC3 supplementation, in accordance with the present disclosure.

FIG. 1K shows the relative percentage of eosinophils (CD11c⁻, CD11b⁺,SiglecF⁺ cells) in lung tissue obtained from three independent studiesin NT mice, CRA treated mice, and mice treated with CRA and an oralsupplementation containing TC3, in accordance with the presentdisclosure.

FIG. 1L shows the relative percentage of neutrophils (CD11b⁻,Gr-1⁺cells) in lung tissue obtained from three independent studies in NTmice, CRA treated mice, and mice treated with CRA and an oral TC3supplementation, in accordance with the present disclosure.

FIG. 2A shows the relative percentage of mast cells (CD49b⁺, CD193⁺,FcεRI⁺, c-kit⁺ cells) in lung tissue obtained from six mice cohortstreated with either PBS, CRA, TC3 (ATCC, frozen), TC3 (WT, frozen), TC3(ATCC, live), or TC3 (WT, live), in accordance with the presentdisclosure.

FIG. 2B shows the relative percentage of basophils (CD49b⁺, CD123⁺,FcεRI⁺ cells) in lung tissue obtained from six mice cohorts treated witheither PBS, CRA, TC3 (ATCC, frozen), TC3 (WT, frozen), TC3 (ATCC, live),or TC3 (WT, live), in accordance with the present disclosure.

FIG. 2C shows the relative percentage of eosinophils (CD11c⁻, CD11b⁺,SiglecF⁺ cells) in lung tissue obtained from six mice cohorts treatedwith either PBS, CRA, TC3 (ATCC, frozen), TC3 (WT, frozen), TC3 (ATCC,live), or TC3 (WT, live), in accordance with the present disclosure.

FIG. 2D shows the relative percentage of neutrophils (CD11b⁺, Gr-1⁺cells) in lung tissue obtained from six mice cohorts treated with eitherPBS, CRA, TC3 (ATCC, frozen), TC3 (WT, frozen), TC3 (ATCC, live), or TC3(WT, live), in accordance with the present disclosure.

FIG. 2E shows the relative percentage of alveolar macrophages (CD11c⁺,CD11b⁻, CD64⁺, SiglecF⁺ cells) in lung tissue obtained from six micecohorts treated with either PBS, CRA, TC3 (ATCC, frozen), TC3 (WT,frozen), TC3 (ATCC, live), or TC3 (WT, live), in accordance with thepresent disclosure.

FIG. 2F shows the relative percentage of T_(H)2 cells (CD3⁺, CD4⁺,GATA-3⁺ cells) in lung tissue obtained from six mice cohorts treatedwith either PBS, CRA, TC3 (ATCC, frozen), TC3 (WT, frozen), TC3 (ATCC,live), or TC3 (WT, live), in accordance with the present disclosure.

FIG. 2G shows the relative percentage of type 17 T helper (T_(H)17)cells (CD3⁺, CD4⁺, RORγt⁺, IL-17A⁺ cells) in lung tissue obtained fromsix mice cohorts treated with either PBS, CRA, TC3 (ATCC, frozen), TC3(WT, frozen), TC3 (ATCC, live), or TC3 (WT, live), in accordance withthe present disclosure.

FIG. 211 shows the relative percentage of T_(reg) cells (CD4⁺, CD127⁻,CD25⁺, Foxp3⁺ cells) in lung tissue obtained from six mice cohortstreated with either PBS, CRA, TC3 (ATCC, frozen), TC3 (WT, frozen), TC3(ATCC, live), or TC3 (WT, live), in accordance with the presentdisclosure.

FIG. 2I shows the relative percentage of CRA-specific type T₂H cells(CD3⁺, CD4⁺, IL-4⁺, GATA-3⁺ cells) in lung tissue obtained from six micecohorts treated with either PBS, CRA, TC3 (ATCC, frozen), TC3 (WT,frozen), TC3 (ATCC, live), or TC3 (WT, live), in accordance with thepresent disclosure.

FIG. 2J shows the relative percentage of follicular B helper T (T_(FH))cells (CD3⁺, CD4⁺, CXCR5⁺, PD-1⁺ cells) in lung tissue obtained from sixmice cohorts treated with either PBS, CRA, TC3 (ATCC, frozen), TC3 (WT,frozen), TC3 (ATCC, live), or TC3 (WT, live), in accordance with thepresent disclosure.

FIG. 2K shows the relative percentage of T_(reg) cells (CD4⁺, CD127⁻,CD25⁺, Foxp3⁺ cells) in medullary lymph node (mLN) tissue obtained fromsix mice cohorts treated with either PBS, CRA, TC3 (ATCC, frozen), TC3(WT, frozen), TC3 (ATCC, live), or TC3 (WT, live), in accordance withthe present disclosure.

FIG. 2L shows the ratio of B regulatory (B_(reg)) cells to B regulatory1 (B_(R)1) cells (CD19⁺, CD5⁺, CD1d⁺ cells) in lung tissue obtained fromsix mice cohorts treated with either PBS, CRA, TC3 (ATCC, frozen), TC3(WT, frozen), TC3 (ATCC, live), or TC3 (WT, live), in accordance withthe present disclosure.

FIG. 2M shows the relative percentage of germinal center (GC) B cells(CD19⁺, CD95⁺, GL7⁺cells) in lung tissue obtained from six mice cohortstreated with either PBS, CRA, TC3 (ATCC, frozen), TC3 (WT, frozen), TC3(ATCC, live), or TC3 (WT, live), in accordance with the presentdisclosure.

FIG. 2N shows the relative percentage of plasma cells (CD19⁻, CD138⁺,CD93⁺cells) in lung tissue obtained from six mice cohorts treated witheither PBS, CRA, TC3 (ATCC, frozen), TC3 (WT, frozen), TC3 (ATCC, live),or TC3 (WT, live), in accordance with the present disclosure.

FIG. 2O shows the IgE concentration (in ng/mL) values in mouse plasmaobtained from six mice cohorts treated with either PBS, CRA, TC3 (ATCC,frozen), TC3 (WT, frozen), TC3 (ATCC, live), or TC3 (WT, live), inaccordance with the present disclosure.

FIG. 2P shows the concentration ratio of CRA-specific IgE toCRA-specific IgG3 in mouse plasma obtained from six mice cohorts treatedwith either PBS, CRA, TC3 (ATCC, frozen), TC3 (WT, frozen), TC3 (ATCC,live), or TC3 (WT, live), in accordance with the present disclosure.

FIG. 2Q shows the histamine concentration values (in ng/mL) in mouseplasma obtained from six mice cohorts treated with either PBS, CRA, TC3(ATCC, frozen), TC3 (WT, frozen), TC3 (ATCC, live), or TC3 (WT, live),in accordance with the present disclosure.

FIG. 2R shows the concentration values of CRA-specific IgE (determinedby measuring the optical density at 450 nm) in mouse plasma obtainedfrom six mice cohorts treated with either PBS, CRA, TC3 (ATCC, frozen),TC3 (WT, frozen), TC3 (ATCC, live), or TC3 (WT, live), in accordancewith the present disclosure.

FIG. 2S shows the concentration values of CRA-specific IgG3 (determinedby measuring the optical density at 450 nm) in mouse plasma obtainedfrom six mice cohorts treated with either PBS, CRA, TC3 (ATCC, frozen),TC3 (WT, frozen), TC3 (ATCC, live), or TC3 (WT, live), in accordancewith the present disclosure.

FIG. 2T shows the concentration values of IL-4 (in pg/mL) in lung tissueobtained from six mice cohorts treated with either PBS, CRA, TC3 (ATCC,frozen), TC3 (WT, frozen), TC3 (ATCC, live), or TC3 (WT, live), inaccordance with the present disclosure.

FIG. 2U shows the concentration values of IL-13 (in pg/mL) in lungtissue obtained from six mice cohorts treated with either PBS, CRA, TC3(ATCC, frozen), TC3 (WT, frozen), TC3 (ATCC, live), or TC3 (WT, live),in accordance with the present disclosure.

FIG. 3 shows a phylogenetic tree generated using whole genome comparisonof A. muciniphila strains. AM-ST7 is framed in black.

FIG. 4 shows a single nucleotide polymorphism (SNP) tree comparing thethree A. muciniphila strains AM-ST7_1, AM-ST7_2, and AM-ST7_3 that aregenetic variants of AM-ST7 with other A. muciniphila strains. The datashows that the three AM-ST7 variants are closely related.

FIG. 5 shows a single nucleotide polymorphism (SNP) tree comparing thethree A. muciniphila strains AM-ST7_1, AM-ST7_2, and AM-ST7_3 that aregenetic variants of AM-ST7 with other A. muciniphila strains. The datashows that the three AM-ST7 variants are closely related.

FIG. 6 shows a table summarizing comparative SNP data between various A.muciniphila strains.

FIG. 7 shows a general outline of a MicroSEQ ID protocol.

FIG. 8 and FIG. 9 show phylogenetic trees generated using whole genomecomparison of Faecalibacterium prausnitzii strains. Faecalibacteriumprausnitzii ST38 is framed in black.

FIG. 10 and FIG. 11 show phylogenetic trees generated using whole genomecomparison of Lactobacillus crispatus strains. Lactobacillus crispatusST100 (LC-ST100) is framed in black.

FIG. 12 shows a snapshot of the ELSD chromatogram of fraction M of A.muciniphila naïve medium (top) and A. muciniphila cell extract (bottom),with signal peaks corresponding to compounds 1-3 (which chemicalstructures are shown in FIG. 13 ).

FIG. 13 shows proposed structures of phosphatidylcholine precursor orphosphatidylcholine-like compounds 1-3.

FIG. 14 shows a summary table of antibiotic resistance results of thethree strains A. muciniphila ST7, Faecalibacterium prausnitzii ST38, andLactobacillus crispatus ST100 (* denotes values as recommended by theClinical and Laboratory Standards Institute). Antibiotic resistance wasdetermined using the broth solution method.

FIG. 15A shows that the A. muciniphila strains AM 2, AM 3, AM 5, AM 6,and AM 7 produced comparable amounts of acetate than the ATCC referencestrain, with AM 3 and AM 7 on the lower end.

FIG. 15B shows that the F. prausnitzii strains FP 12, FP 13, FP 14, FP15, and FP 16 produced higher amounts of acetate than the ATCC referencestrain. Surprisingly, the strains FP 15 and FP 16 produced more thantwice the amount of acetate compared to the ATCC reference strain.

FIG. 15C shows that the F. prausnitzii strains FP 12, FP 13, FP 14, FP15, and FP 16 produce significantly higher amounts of isobutyratecompared to the ATCC reference strain or the CMC media control.

FIG. 16 illustrates an example of a timeline and setup for experimentalstudies using an allergic airway inflammation mouse model to evaluatethe protective effect of phosphatidylcholines orphosphatidylcholine-derived compounds producing intestinal bacterialstrains on allergic airway inflammation.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The present disclosure provides methods and compositions comprisingmicrobial compositions, such as therapeutic microbial consortia. Themicrobial compositions described herein can comprise one or moredifferent bacterial species (e.g., Akkermansia sp., Faecalibacteriumsp., Lactobacillus sp., and/or those listed in Table 1) and/or one ormore different bacterial strains (e.g., any one or more of those listedin Table 1 (strains listed as isolated numbers) and/or Table 2 (strainslisted as isolated numbers and strain numbers)). Such microbialcompositions can have beneficial and/or therapeutic properties and hencecan be used to prevent and/or treat a broad spectrum of diseases ordisorders in a subject (e.g., a rodent or a human) upon administrationto said subject. The microbial compositions described herein can beadministered using various administration routes, includingadministration as an oral dosage form (e.g., a capsule, a tablet, anemulsion, suspension, etc.).

A microbial composition described herein can comprise any one or more ofthe bacterial species Akkermansia sp., Faecalibacterium sp.,Lactobacillus sp., or any combination thereof. A microbial compositioncan comprise strains A. muciniphila ST7, F. prausnitzii ST38, or L.crispatus ST100, or any combination thereof. A microbial composition canbe a synergistic bacterial consortium. Synergistic effects of such aconsortium can include increased therapeutic efficacy in a subjectand/or beneficial long-term health effects (e.g., prevention of diseasesor conditions) for said subject. A microbial composition of the presentdisclosure can comprise one or more bacterial species and/or strainsthat can produce one or more beneficial and/or therapeutically effectivecompounds. Such compounds can have anti-inflammatory effects. Thecompositions and methods described herein can comprise one or morebacterial cells of one or more bacterial species or strains that producebeneficial fatty acids including short-chain fatty acids (SCFAs),saturated and unsaturated fatty acids such as omega-3 and/or omega-6fatty acids (e.g., linolenic acid), and/or effect or alter a metabolismof such fatty acids. The compositions and methods described herein cancomprise one or more bacterial cell of one or more bacterial species orstrains that produce and/or affect or alter the metabolism ofphospholipids and/or derivatives thereof. Such phospholipid and/orderivative thereof can be a phosphatidylcholine and/or a derivativethereof. The present disclosure provides one or more species or strainsof Akkermansia sp. that can produce one or more phospholipids (e.g.,phosphatidylcholine(s) and/or derivative(s) thereof) and/or derivativesthereof. The Akkermansia strain that produces one or more phospholipidscan be A. muciniphila ST7. At least one of the phospholipids that can beproduced by A. muciniphila ST7 can be a phosphatidylcholine and/or aderivative thereof. Such compounds can have structural similarity withcompounds 1-3 shown in FIG. 14 .

The therapeutic consortia described herein can be used to treat and/orprevent diseases and conditions such as dysbiosis, inflammation (e.g.,chronic and/or allergic inflammation), autoimmune disorders, infections,and/or cancer.

Whenever the term “at least,” “greater than,” or “greater than or equalto” precedes the first numerical value in a series of two or morenumerical values, the term “at least,” “greater than” or “greater thanor equal to” applies to each of the numerical values in that series ofnumerical values. For example, greater than or equal to 1, 2, or 3 isequivalent to greater than or equal to 1, greater than or equal to 2, orgreater than or equal to 3.

Whenever the term “no more than,” “less than,” or “less than or equalto” precedes the first numerical value in a series of two or morenumerical values, the term “no more than,” “less than,” or “less than orequal to” applies to each of the numerical values in that series ofnumerical values. For example, less than or equal to 3, 2, or 1 isequivalent to less than or equal to 3, less than or equal to 2, or lessthan or equal to 1.

As used herein, a substance is “pure” or “substantially pure” if it issubstantially free of other components. The terms “purify,” “purifying”and “purified”, when applied to a bacterium, can refer to a bacteriumthat has been separated from at least some of the components with whichit was associated either when initially produced or generated (e.g.,whether in nature or in an experimental setting), or during any timeafter its initial production. A bacterium or a bacterial population maybe considered purified if it is isolated at or after production, such asfrom a material or environment containing the bacterium or bacterialpopulation, or by passage through culture, and a purified bacterium orbacterial population may contain other materials up to at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or above about 90% and still be considered“isolated.” Purified bacteria and bacterial populations can be more thanat least about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than at least about 99% pure by weight (w/w). In the instance ofmicrobial compositions provided herein, the one or more bacterial types(species or strains) present in the composition can be independentlypurified from one or more other bacteria produced and/or present in thematerial or environment containing the bacterial type. Microbialcompositions and the bacterial components thereof are generally purifiedfrom residual habitat products.

An isolated bacterium may have been (1) separated from at least some ofthe components with which it was associated when initially obtained(whether in nature or in an experimental setting), and/or (2) produced,prepared, purified, and/or manufactured by the hand of man, e.g. usingartificial culture conditions such as (but not limited to) culturing ona plate and/or in a fermenter. Isolated bacteria can include thosebacteria that are cultured, even if such cultures are not monocultures.Isolated bacteria can be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, or more of the other components with which they were initiallyassociated. Isolated bacteria can be more than about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or more than about 99% pure. Abacterial population of a biological sample provided herein can compriseone or more bacteria, which may then be isolated from such sample.Isolated bacteria may be provided in a form that is not naturallyoccurring.

As used herein, the terms “effective amount” and “effective dosage” canbe used interchangeably, and generally refer to an amount necessary toproduce a desired physiologic response (e.g., reduction of inflammation,infection, or dysbiosis). Effective amounts and schedules foradministering a therapeutic consortium may be determined empirically.The dosage ranges for administration can be large enough to produce thedesired effect in which one or more symptoms of the disease or disorderare affected (e.g., reduced or delayed). The dosage may not be so largeas to cause substantial adverse side effects, such as cross-reactions,anaphylactic reactions, and the like. Generally, the dosage will varywith the age, condition, sex, type of disease, the extent of the diseaseor disorder, route of administration, or whether other drugs areincluded in the regimen, and can be determined. The dosage can beadjusted in the event of any contraindications. Dosages can vary and canbe administered in one or more dose administrations daily, for one orseveral days. For example, for the given parameter, an effective amountwill show an increase or decrease of at least about 5%, 10%, 15%, 20%,25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can alsobe expressed as “-fold” increase or decrease. For example, atherapeutically effective amount can have at least about a 1.2-fold,1.5-fold, 2-fold, 5-fold, or more effect over a control. Forprophylactic use, a therapeutically effective amount of the microbialcompositions described herein are administered to a subject prior to orduring early onset (e.g., upon initial signs and symptoms of anautoimmune disease).

As used herein, the terms “subject,” “patient,” “individual,” etc. canbe generally interchanged. In addition, an individual described as a“patient” may not have a given disease, but may be merely seekingmedical advice As used herein, “treating” or treatment of a condition,disease or disorder or symptoms associated with a condition, disease ordisorder refers to an approach for obtaining beneficial results,including clinical results. Beneficial clinical results can include, butare not limited to, alleviation or amelioration of one or more symptomsor conditions, diminishment of extent of condition, disorder or disease,stabilization of the state of condition, disorder or disease, preventionof development of condition, disorder or disease, prevention of spreadof condition, disorder or disease, delay or slowing of condition,disorder or disease progression, delay or slowing of condition, disorderor disease onset, amelioration or palliation of the condition, disorderor disease state, and remission, whether partial or total. “Treating”can also mean prolonging survival of a subject beyond that expected inthe absence of treatment. “Treating” can also mean inhibiting theprogression of the condition, disorder or disease, slowing theprogression of the condition, disorder or disease temporarily, althoughin some instances, it can involve halting the progression of thecondition, disorder or disease permanently. As used herein the termstreatment, treat, or treating refers to a method of reducing the effectsof one or more symptoms of a disease or condition characterized byexpression of the protease or symptom of the disease or conditioncharacterized by expression of the protease. Thus, the herein disclosedmethods or treatments can refer to at least about a 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of anestablished disease, condition, or symptom of the disease or condition(e.g., inflammation, infection, or dysbiosis). For example, a method fortreating a disease is considered to be a treatment if there is a 10%reduction in one or more symptoms of the disease in a subject ascompared to a control. Thus, the reduction can be at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reductionin between about 10% and about 100% as compared to native or controllevels. Further, as used herein, references to decreasing, reducing, orinhibiting include a change of at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or greater as compared to a control level and suchterms can include but do not necessarily include complete elimination.Compositions comprising a defined microbial compositions can beadministered to the gastrointestinal tract of a subject by nasoduodenalcatheter, by enema, or by endoscopy, enteroscopy, or colonoscopy ororally in a consumable capsule, pill, solution, suspension, etc. Atherapeutic microbial composition can be diluted in a suitable excipient(e.g., comprising saline solution, buffer, etc.).

As described herein, the terms “disease” and “condition” can be usedinterchangeably herein and generally refer to a state of being or healthstatus of a patient or subject capable of being diagnosed and/or treatedwith a kit, method and/or composition disclosed herein. A disease can bean inflammatory disease, an infectious disease, or an autoimmunedisease. A disease can be or can be associated with a microbiome of asubject, such as an intestinal microbiome. A disease can be a dysbiosis,such as gut dysbiosis.

As described herein, the term “dysbiosis” can mean a difference in thegastrointestinal microbiota compared to a healthy or general population.Dysbiosis can comprise a difference in gastrointestinal microbiotacommensal species diversity compared to a healthy or general population.Dysbiosis can also comprise a decrease of beneficial microorganismsand/or increase of pathobionts (pathogenic or potentially pathogenicmicroorganisms) and/or decrease of overall microbiota species diversity.Many factors can harm the beneficial members of the intestinalmicrobiota leading to dysbiosis, including (but not limited to)antibiotic use, psychological and physical stress, radiation, anddietary changes. Dysbiosis can comprise and/or promote the overgrowth ofa bacterial opportunistic pathogen such as Enterococcus faecalis,Enterococcusfaecium, or Clostridium difficile. A dysbiosis can comprisea reduced amount (absolute number or proportion of the total microbialpopulation) of bacterial or fungal cells of a species or genus (e.g., atleast about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more lower) compared to a healthysubject (e.g., a corresponding subject who does not have an inflammatorydisease, an infection, and who has not been administered an antibioticwithin about 1, 2, 3, 4, 5,or 6 months, and/or compared to a healthy orgeneral population). The dysbiosis comprises an increased amount(absolute number or proportion of the total microbial population) ofbacterial or fungal cells within a species or genus (e.g., at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or more higher) compared to a healthysubject (e.g., a corresponding subject who does not have an inflammatorydisease, an infection, and who has not been administered an antibioticwithin about 1, 2, 3, 4, 5,or 6 months, and/or compared to a healthy orgeneral population). A subject who comprises a gastrointestinalinfection, gastrointestinal inflammation, diarrhea, colitis, or who hasreceived an antibiotic within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10weeks can be deemed to comprise dysbiosis. An impaired microbiota cancomprise small intestinal bacterial or fungal overgrowth. Antibioticadministration (e.g., systemically, such as by intravenous injection ororally) can be a common and significant cause of major alterations inthe normal microbiota. Thus, as used herein, the term“antibiotic-induced dysbiosis can refer to dysbiosis caused by orfollowing the administration of an antibiotic. Dysbiosis can beassociated with various diseases and conditions such as inflammation(e.g., chronic and/or allergic inflammation), auto-immune disorders,infections, and/or cancer. Thus, a subject suffering from dysbiosis canhave an inflammatory disease (e.g., chronic and/or allergic inflammatorydisease), auto-immune disorders, infections, and/or cancer.

As described herein, the term “diagnosis” generally refers to a relativeprobability that a disease (e.g., an autoimmune, inflammatoryautoimmune, cancer, infectious, immune, dysbiosis, etc.) is present in asubject. Similarly, the term “prognosis” generally refers to a relativeprobability that a certain future outcome may occur in the subject withrespect to a disease state.

As described herein, the terms “biological sample” or “sample” can beused interchangeably herein and generally refer to materials obtainedfrom or derived from a subject (e.g., a human). A biological sample caninclude sections of tissues such as biopsy and autopsy samples, andfrozen sections taken for histological purposes. Such samples caninclude bodily fluids such as blood and blood fractions or products(e.g., serum, plasma, platelets, red blood cells, and the like), fecesand feces fractions or products (e.g., fecal water, such as but notlimited to fecal water separated from other fecal components and solidsby methods such as centrifugation and filtration) sputum, tissue,cultured cells (e.g., primary cultures, explants, and transformedcells), stool, urine, synovial fluid, joint tissue, synovial tissue,synoviocytes, fibroblast-like synoviocytes, macrophage-likesynoviocytes, immune cells, hematopoietic cells, fibroblasts,macrophages, dendritic cells, T-cells, etc. A sample can be obtainedfrom a eukaryotic organism, such as a mammal such as a primate e.g.,chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat,mouse; rabbit; or a bird; reptile; or fish.

As described herein, the abbreviation “sp.” for species can mean atleast one species (e.g., 1, 2, 3, 4, 5, or more species) of theindicated genus. The abbreviation “spp.” for species can mean 2 or morespecies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the indicatedgenus. Generally, a first and a second bacterium may be differentiatedat various taxonomic leveles, for example, the genus level (e.g.,Akkermansia sp.), the species level (e.g., Akkermansia muciniphila), thestrain level (e.g., Akkermansia muciniphila ST7, or AM-ST7), and/or avariant (e.g., a genetic variant) level (e.g., AM-ST7_1), or by anyother taxonomic method. The methods and compositions provided herein cancomprise a single species within an indicated genus or indicated genera,or 2 or more (e.g., a plurality comprising more than 2) species withinan indicated genus or indicated genera. Furthermore, the methods andcompositions described herein can comprise one or more strains of anindicated species. A strain (e.g., a Akkermansia muciniphila ST7 orAM-ST7) of the present disclosure can comprise one or more geneticvariants (e.g., AM-ST7_1, AM-ST7_2, and/or AM-ST7_3). Thus, the methodsand compositions of the present disclosure can comprise one or moredifferent variants of an indicated strain. Such genetic strain variantscan have a high genetic similarity to each other as exemplified in FIG.4 -FIG. 6 for the variants AM-ST7_1, AM-ST7_2, and AM-ST7_3.

As described herein, the terms “bacterial composition”, “bacterialpopulation”, and “bacterial consortium” can be used interchangeablyherein.

As described herein, the term “derivative” in the context of chemicalcompounds generally refers to any chemical derivative of such compound,including any isomer, conformer, or otherwise structurally, chemically,or biologically related compounds. For example, the term“phosphatidylcholine derivative” generally refers to anyphosphatidylcholine derivative, any phosphatidylcholine-like compound,any phosphatidylcholine isomer, conformer, or otherwise structurally,chemically, or biologically related compounds to the compound class ofphosphatidylcholines. Moreover, the terms “phosphatidylcholinederivative” and “phosphatidylcholine-like compound” can be usedinterchangeably herein. In an example, a bacterial strain (e.g., aAkkermansia muciniphila strain) of the present disclosure can produceone or more phosphatidylcholine-like compounds. Such compounds can havea therapeutic effect, and may be used to prevent and/or treat a diseaseor condition in a subject.

The present disclosure provides for the identification,characterization, and use of bacterial consortia for the treatment ofdiseases or disorders that include but are not limited to dysbiosis,asthma, allergy, infections, and inflammatory diseases. Advances ingenomics and next-generation sequencing can allow for identification andcharacterization of specific bacterial strains that inhabit the humangut. A presence or an absence of one or more of such bacterial strainsin a subject can be associated with a disease or condition, such as adysbiotic condition, an asthmatic condition, an allergic condition, aninfection, or an inflammatory condition in a subject. Thecharacterization and stratification of bacterial consortia comprisingsuch bacterial strains (e.g., those listed in Table 1) may provide aplatform for the identification of compositions comprising bacteria thatcan act synergistically to treat and/or prevent one or more of theaforementioned conditions.

Microbial Consortia

Generally, a microbial consortium of the present disclosure can compriseone or more genera, species, strains, and/or strain variants belongingto the phyla Verrucomicrobia, Firmicutes, Proteobacteria,Actinobacteria, and/or Bacteroidetes, or any combination thereof. Amicrobial consortium can comprise bacteria belonging to one or more ofthe genera Faecalibacterium sp., Akkermansia sp, Lactobacillus sp., orany combination thereof.

A microbial consortium can comprise less than about 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 different species ofbacteria. A microbial consortium can comprise less than about 20different species of bacteria. The composition can include less thanabout 20 different species of bacteria. A microbial consortium cancomprise less than about 15 different species of bacteria. A microbialconsortium can comprise less than 15 different species of bacteria. Amicrobial consortium can comprise less than about 10 different speciesof bacteria. A microbial consortium can comprise less than 10 differentspecies of bacteria. A microbial consortium can comprise less than about9 different species of bacteria. A microbial consortium can compriseless than 9 different species of bacteria. A microbial consortium cancomprise less than about 8 different species of bacteria. A microbialconsortium can comprise less than 8 different species of bacteria. Amicrobial consortium can comprise less than about 7 different species ofbacteria. A microbial consortium can comprise less than 7 differentspecies of bacteria. A microbial consortium can comprise than about 6different species of bacteria. A microbial consortium can comprise than6 different species of bacteria. A microbial consortium can compriseless than about 5 different species of bacteria. A microbial consortiumcan comprise less than 5 different species of bacteria. A microbialconsortium can comprise less than about 4 different species of bacteria.A microbial consortium can comprise less than 4 different species ofbacteria. A microbial consortium can comprise less than about 3different species of bacteria. A microbial consortium can comprise lessthan 3 different species of bacteria. A microbial consortium cancomprise less than about 2 different species of bacteria. A microbialconsortium can comprise less than 2 different species of bacteria.

The present disclosure provides and compositions and methods that cancomprise microbial compositions comprising, consisting essentially of,or consisting of one or more (i) Lactobacillus strain(s), one or more(ii) Faecalibacterium strain(s), one or more (iii) Akkermansiastrain(s), and/or one or more (iv) Bifidobacterium strain(s) fortreating a disease or disorder. The compositions can comprise at leastone strain of Lactobacillus johnsonii or Lactobacillus crispatus, atleast one strain of Faecalibacterium prausnitzii, at least one strain ofAkkermansia muciniphila, and in some cases at least one strain ofBifidobacterium longum or another suitable Bifidobacterium.Lactobacillus johnsonii and Lactobacillus crispatus is a species in thegenus of Lactobacillus that belongs to the phylum Firmicutes.Faecalibacterium prausnitzii is the only known species in theFaecalibacterium genus, and it belongs to the phylum Firmicutes.Akkermansia muciniphila is a species within the genus of Akkermansiathat belongs to the phylum Verruccomicrobia. Bifidobacteria is a genusof gram-positive bacteria that belong to the phylum Actinobacteria.

The present disclosure provides compositions and methods that cancomprise microbial compositions comprising a Lactobacillus species thatcan be Lactobacillus johnsonii or Lactobacillus crispatus. TheLactobacillus species can be Lactobacillus johnsonii, Lactobacillusrhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri , Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Laclococcus garvieae, or Lactococcus lactis, or acombination thereof.

The present disclosure provides compositions and methods that cancomprise microbial compositions comprising an Akkermansia species thatis Akkermansia muciniphila. The Akkermansia species can be Akkermansiamucimphila or Akkermansia glycamphila, or a combination thereof.

The present disclosure provides compositions and methods where theBifidobacterium species can be Bifidobacterium faecale. TheBifidobacterium species can be Bifidobacterium angulatum,Bifidobacterium animalis, Bifidobacterium asteroides; Bifidobacteriumbifidum; Bifidobacterium boum; Bifidobacterium breve, Bifidobacteriumcatenulatum, Bifidobacterium choerinum, Bifidobacterium coryneforme,Bifidobacterium cuniculi, Bifidobacterium dentium, Bifidobacteriumgallicum, Bifidobacterium gallinarum, Bifidobacterium indicum,Bifidobacterium longum, Bifidobacterium longum infantis, Bifidobacteriummagnum, Bifidobacterium merycicum, Bifidobacterium minimum,Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum,Bifidobacterium psychraerophilum, Bifidobacterium pullorum,Bifidobacterium ruminantium, Bifidobacterium saeculare, Bifidobacteriumscardovii, Bifidobacterium simiae, Bifidobacterium stercoris,Bifidobacterium subtile, Bifidobacterium thermacidophilum,Bifidobacterium thermophilum, or Bifidobacterium urinalis, or acombination thereof.

The present disclosure provides microbial compositions that can compriseat least one Akkermansia species. A microbial composition can compriseat least one Faecalibacterium species. A microbial composition cancomprise at least one Lactobacillus species. A microbial composition cancomprise at least one Akkermansia species and at least oneFaecalibacterium species. A microbial composition can comprise at leastone Akkermansia species and at least one Lactobacillus species(Lactobacillus sp.). A microbial composition can comprise at least oneFaecalibacterium species (Faecalibacterium sp.) and at least oneLactobacillus species. The present disclosure provides microbialcompositions that can comprise at least one Akkermansia species, atleast one Faecalibacterium species and at least one Lactobacillusspecies (e.g., any of those listed below in Table 1, second to the leftcolumn).

The compositions and methods described herein can comprise one or morebacterial populations that can be described and characterized on aspecies level (e.g., Akkermansia mucimphila). The compositions andmethods described herein can also comprise one or more bacterialpopulations that can be described and characterized on a strain level(e.g., Akkermansia mucimphila ST7). The compositions and methodsdescribed herein can also comprise one or more bacterial populationsthat can be described and characterized on a genetic variant level(e.g., Akkermansia mucimphila ST7_1), wherein the genetic variants of anindicated strain can be closely related, e.g., as those described inFIG. 4 and FIG. 5 .

The present disclosure provides compositions and methods that cancomprise one or more of the bacterial species and/or strains shown asisolate numbers 1-251 described in Table 1 below, or any combination ofsuch strains (i.e., each isolate number of Table 1 corresponds to anisolated bacterial strain).

TABLE 1 Examples of Strains Used in Therapeutic Consortia Isolate NumberSpecies Source Isolation Media 1 Akkermansia muciniphila Human - fecalMucin Minimal Media 2 Akkermansia muciniphila Human - fecal MucinMinimal Media 3 Akkermansia muciniphila Human - fecal Mucin MinimalMedia 4 Akkermansia muciniphila Human - fecal Mucin Minimal Media 5Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 6Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 7Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 8Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 9Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 10Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 11Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 12Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 13Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 14Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 15Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 16Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 17Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 18Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 19Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 20Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 21Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 22Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 23Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 24Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 25Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 26Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 27Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 28Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 29Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 30Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 31Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 32Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 33Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 34Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 35Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 36Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 37Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 38Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 39Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 40Akkermansia muciniphila Mus musculus - fecal Mucin Minimal Media 41Anaerostipes hadrus Human - fecal Yeast Fatty Acid Casitone Media 42Anaerotignum lactatifermentans Human - fecal Yeast Fatty Acid CasitoneMedia 43 Bacteroides caccae Human - fecal Yeast Fatty Acid CasitoneMedia 44 Bacteroides caccae Human - fecal Yeast Fatty Acid CasitoneMedia 45 Bacteroides dorei Human - fecal Yeast Fatty Acid Casitone Media46 Bacteroides dorei Human - fecal Yeast Fatty Acid Casitone Media 47Bacteroides dorei Human - fecal Mucin Minimal Media 48 Bacteroidesfaecis Human - fecal Mucin Minimal Media 49 Bacteroides faecis Human -fecal Mucin Minimal Media 50 Bacteroides faecis Human - fecal YeastFatty Acid Casitone Media 51 Bacteroides faecis Human - fecal YeastFatty Acid Casitone Media 52 Bacteroides faecis Human - fecal YeastFatty Acid Casitone Media 53 Bacteroides faecis Human - fecal YeastFatty Acid Casitone Media 54 Bacteroides faecis Human - fecal YeastFatty Acid Casitone Media 55 Bacteroides faecis Human - fecal YeastFatty Acid Casitone Media 56 Bacteroides faecis Human - fecal YeastFatty Acid Casitone Media 57 Bacteroides faecis Human - fecal YeastFatty Acid Casitone Media 58 Bacteroides finegoldii Human - fecal YeastFatty Acid Casitone Media 59 Bacteroides fragilis Human - fecal YeastFatty Acid Casitone Media 60 Bacteroides fragilis Human - fecal YeastFatty Acid Casitone Media 61 Bacteroides fragilis Human - fecal YeastFatty Acid Casitone Media 62 Bacteroides fragilis Human - fecal YeastFatty Acid Casitone Media 63 Bacteroides fragilis Human - fecal YeastFatty Acid Casitone Media 64 Bacteroides fragilis Human - fecal YeastFatty Acid Casitone Media 65 Bacteroides fragilis Human - fecal YeastFatty Acid Casitone Media 66 Bacteroides fragilis Human - fecal MucinMinimal Media 67 Bacteroides fragilis Human - fecal Mucin Minimal Media68 Bacteroides fragilis Human - fecal Mucin Minimal Media 69 Bacteroidesfragilis Human - fecal Yeast Fatty Acid Casitone Media 70 Bacteroidesfragilis Human - fecal Yeast Fatty Acid Casitone Media 71 Bacteroidesfragilis Human - fecal Yeast Fatty Acid Casitone Media 72 Bacteroidesfragilis Human - fecal Yeast Fatty Acid Casitone Media 73 Bacteroidesintestinalis Human - fecal Yeast Fatty Acid Casitone Media 74Bacteroides stercoris Human - fecal Yeast Fatty Acid Casitone Media 75Bacteroides stercoris Human - fecal Yeast Fatty Acid Casitone Media 76Bacteroides stercoris Human - fecal Yeast Fatty Acid Casitone Media 77Bacteroides stercoris Human - fecal Yeast Fatty Acid Casitone Media 78Bacteroides stercoris Human - fecal Yeast Fatty Acid Casitone Media 79Bacteroides stercoris Human - fecal Yeast Fatty Acid Casitone Media 80Bacteroides stercoris Human - fecal Yeast Fatty Acid Casitone Media 81Bacteroides stercoris Human - fecal Yeast Fatty Acid Casitone Media 82Bacteroides thetaiotaomicron Human - fecal Yeast Fatty Acid CasitoneMedia 83 Bacteroides thetaiotaomicron Human - fecal Yeast Fatty AcidCasitone Media 84 Bacteroides thetaiotaomicron Human - fecal Yeast FattyAcid Casitone Media 85 Bacteroides thetaiotaomicron Human - fecal YeastFatty Acid Casitone Media 86 Bacteroides thetaiotaomicron Human - fecalYeast Fatty Acid Casitone Media 87 Bacteroides thetaiotaomicron Human -fecal Yeast Fatty Acid Casitone Media 88 Bacteroides thetaiotaomicronMus musculus - fecal Mucin Minimal Media 89 Bacteroides thetaiotaomicronHuman - fecal Yeast Fatty Acid Casitone Media 90 Bacteroidesthetaiotaomicron Human - fecal Mucin Minimal Media 91 Bacteroidesthetaiotaomicron Human - fecal Yeast Fatty Acid Casitone Media 92Bacteroides uniformis Human - fecal Yeast Fatty Acid Casitone Media 93Bacteroides uniformis Human - fecal Yeast Fatty Acid Casitone Media 94Bacteroides uniformis Human - fecal Yeast Fatty Acid Casitone Media 95Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 96Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 97Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 98Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 99Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 100Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 101Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 102Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 103Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 104Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 105Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 106Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 107Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 108Bacteroides vulgatus Human - fecal Yeast Fatty Acid Casitone Media 109Bacteroides xylanisolvens Human - fecal Mucin Minimal Media 110Bacteroides xylanisolvens Human - fecal Yeast Fatty Acid Casitone Media111 Bifidobacterium faecale Human - fecal Bifidobacterium SelectiveMedia 112 Bifidobacterium faecale Human - fecal BifidobacteriumSelective Media 113 Bifidobacterium longum Human - fecal BifidobacteriumSelective Media 114 Bifidobacterium stercoris Human - fecalBifidobacterium Selective Media 115 Bifidobacterium stercoris Human -fecal Bifidobacterium Selective Media 116 Blautia faecis Human - fecalYeast Fatty Acid Casitone Media 117 Blautia faecis Human - fecal YeastFatty Acid Casitone Media 118 Blautia gnavus Human - fecal Yeast FattyAcid Casitone Media 119 Blautia luti Human - fecal Yeast Fatty AcidCasitone Media 120 Blautia obeum Human - fecal Yeast Fatty Acid CasitoneMedia 121 Blautia obeum Human - fecal Yeast Fatty Acid Casitone Media122 Blautia obeum Human - fecal Yeast Fatty Acid Casitone Media 123Blautia obeum Human - fecal Yeast Fatty Acid Casitone Media 124 Blautiaobeum Human - fecal Yeast Fatty Acid Casitone Media 125 Blautia obeumHuman - fecal Yeast Fatty Acid Casitone Media 126 Blautia productHuman - fecal Yeast Fatty Acid Casitone Media 127 Blautia productHuman - fecal Yeast Fatty Acid Casitone Media 128 Blautia productHuman - fecal Yeast Fatty Acid Casitone Media 129 Blautia productHuman - fecal Yeast Fatty Acid Casitone Media 130 Blautia productHuman - fecal Yeast Fatty Acid Casitone Media 131 Blautia productHuman - fecal Yeast Fatty Acid Casitone Media 132 Blautia productHuman - fecal Yeast Fatty Acid Casitone Media 133 Blautia productHuman - fecal Mucin Minimal Media 134 Blautia product Human - fecalMucin Minimal Media 135 Blautia product Human - fecal Yeast Fatty AcidCasitone Media 136 Blautia stercoris Human - fecal Yeast Fatty AcidCasitone Media 137 Blautia stercoris Human - fecal Yeast Fatty AcidCasitone Media 138 Blautia torque Human - fecal Yeast Fatty AcidCasitone Media 139 Blautia wexlerae Human - fecal Yeast Fatty AcidCasitone Media 140 Blautia wexlerae Human - fecal Yeast Fatty AcidCasitone Media 141 Blautia wexlerae Human - fecal Mucin Minimal Media142 Blautia wexlerae Human - fecal Yeast Fatty Acid Casitone Media 143Blautia wexlerae Human - fecal Yeast Fatty Acid Casitone Media 144Blautia wexlerae Human - fecal Mucin Minimal Media 145 Blautia wexleraeHuman - fecal Yeast Fatty Acid Casitone Media 146 Collinsellaaerofaciens Human - fecal Yeast Fatty Acid Casitone Media 147Coprococcus comes Human - fecal Yeast Fatty Acid Casitone Media 148Coprococcus comes Human - fecal Yeast Fatty Acid Casitone Media 149Coprococcus comes Human - fecal Yeast Fatty Acid Casitone Media 150Coprococcus comes Human - fecal Yeast Fatty Acid Casitone Media 151Coprococcus comes Human - fecal Yeast Fatty Acid Casitone Media 152Coprococcus comes Human - fecal Yeast Fatty Acid Casitone Media 153Coprococcus comes Human - fecal Yeast Fatty Acid Casitone Media 154Coprococcus eutactus Human - fecal Yeast Fatty Acid Casitone Media 155Dorea formicigenerans Human - fecal Yeast Fatty Acid Casitone Media 156Dorea formicigenerans Human - fecal Yeast Fatty Acid Casitone Media 157Dorea formicigenerans Human - fecal Yeast Fatty Acid Casitone Media 158Dorea longicatena Human - fecal Yeast Fatty Acid Casitone Media 159Dorea longicatena Human - fecal Yeast Fatty Acid Casitone Media 160Dorea longicatena Human - fecal Yeast Fatty Acid Casitone Media 161Eisenbergiella massiliensis Human - fecal Yeast Fatty Acid CasitoneMedia 162 Erysipelatoclostridium ramosum Human - fecal Yeast Fatty AcidCasitone Media 163 Erysipelatoclostridium ramosum Human - fecal YeastFatty Acid Casitone Media 164 Erysipelatoclostridium ramosum Human -fecal Yeast Fatty Acid Casitone Media 165 Erysipelatoclostridium ramosumHuman - fecal Yeast Fatty Acid Casitone Media 166 Erysipelatoclostridiumramosum Human - fecal Yeast Fatty Acid Casitone Media 167Erysipelatoclostridium ramosum Human - fecal Yeast Fatty Acid CasitoneMedia 168 Erysipelatoclostridium ramosum Human - fecal Yeast Fatty AcidCasitone Media 169 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 170 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 171 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 172 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 173 Escherichia.fergusonii Human - fecal Yeast Fatty AcidCasitone Media 174 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 175 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 176 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 177 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 178 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 179 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 180 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 181 Escherichia fergusonii Human - fecal Yeast Fatty AcidCasitone Media 182 Eubacterium hallii Human - fecal Yeast Fatty AcidCasitone Media 183 Eubacterium ventriosum Human - fecal Yeast Fatty AcidCasitone Media 184 Faecalibacterium prausnitzii Human - fecal YeastFatty Acid Casitone Media 185 Faecalibacterium prausnitzii Human - fecalYeast Fatty Acid Casitone Media 186 Faecalibacterium prausnitzii Human -fecal Yeast Fatty Acid Casitone Media 187 Faecalibacterium prausnitziiHuman - fecal Yeast Fatty Acid Casitone Media 188 Faecalibacteriumprausnitzii Human - fecal Yeast Fatty Acid Casitone Media 189Faecalibacterium prausnitzii Human - fecal Yeast Fatty Acid CasitoneMedia 190 Faecalibacterium prausnitzii Human - fecal Yeast Fatty AcidCasitone Media 191 Faecalibacterium prausnitzii Human - fecal YeastFatty Acid Casitone Media 192 Faecalibacterium prausnitzii Human - fecalYeast Fatty Acid Casitone Media 193 Faecalibacterium prausnitzii Human -fecal Yeast Fatty Acid Casitone Media 194 Faecalibacterium prausnitziiHuman - fecal Yeast Fatty Acid Casitone Media 195 Faecalibacteriumprausnitzii Human - fecal Yeast Fatty Acid Casitone Media 196Faecalibacterium prausnitzii Human - fecal Yeast Fatty Acid CasitoneMedia 197 Faecalibacterium prausnitzii Human - fecal Yeast Fatty AcidCasitone Media 198 Faecalibacterium prausnitzii Human - fecal YeastFatty Acid Casitone Media 199 Faecalibacterium prausnitzii Human - fecalYeast Fatty Acid Casitone Media 200 Holdemanella biforme Human - fecalYeast Fatty Acid Casitone Media 201 Holdemanella biforme Human - fecalYeast Fatty Acid Casitone Media 202 Holdemanella biforme Human - fecalYeast Fatty Acid Casitone Media 203 Hungatella effluvia Human - fecalYeast Fatty Acid Casitone Media 204 Hungatella hathewayi Human - fecalYeast Fatty Acid Casitone Media 205 Lachnoclostridium aerotoleransHuman - fecal Yeast Fatty Acid Casitone Media 206 Lachnoclostridiumaerotolerans Human - fecal Yeast Fatty Acid Casitone Media 207Lachnoclostridium aerotolerans Human - fecal Yeast Fatty Acid CasitoneMedia 208 Lachnoclostridium aerotolerans Human - fecal Yeast Fatty AcidCasitone Media 209 Lachnoclostridium bolteae Human - fecal Yeast FattyAcid Casitone Media 210 Lachnoclostridium lavalense Human - fecal YeastFatty Acid Casitone Media 211 Lachnoclostridium symbiosum Human - fecalYeast Fatty Acid Casitone Media 212 Lachnospira pectinoschiza Human -fecal Yeast Fatty Acid Casitone Media 213 Lactobacillus coleohominisHuman - vaginal de Man Rogosa Sharpe Media 214 Lactobacillus crispatusHuman - vaginal de Man Rogosa Sharpe Media 215 Lactobacillus crispatusHuman - vaginal de Man Rogosa Sharpe Media 216 Lactobacillus crispatusHuman - vaginal de Man Rogosa Sharpe Media 217 Lactobacillus gasseri Musmusculus - fecal de Man Rogosa Sharpe Media 218 Lactobacillus jenseniiHuman - vaginal de Man Rogosa Sharpe Media 219 Lactobacillus jenseniiHuman - vaginal de Man Rogosa Sharpe Media 220 Lactobacillus jenseniiHuman - vaginal de Man Rogosa Sharpe Media 221 Lactobacillus jenseniiHuman - vaginal de Man Rogosa Sharpe Media 222 Lactobacillus jenseniiHuman - vaginal de Man Rogosa Sharpe Media 223 Lactobacillus johnsoniiMus musculus - fecal de Man Rogosa Sharpe Media 224 Lactobacillusjohnsonii Mus musculus - fecal de Man Rogosa Sharpe Media 225Lactobacillus johnsonii Mus musculus - fecal de Man Rogosa Sharpe Media226 Lactobacillus johnsonii Mus musculus - fecal de Man Rogosa SharpeMedia 227 Lactonifactor longoviformis Human - fecal Yeast Fatty AcidCasitone Media 228 Longibaculum muris Human - fecal Yeast Fatty AcidCasitone Media 229 Longibaculum muris Human - fecal Yeast Fatty AcidCasitone Media 230 Longibaculum muris Human - fecal Yeast Fatty AcidCasitone Media 231 Longibaculum muris Human - fecal Yeast Fatty AcidCasitone Media 232 Muribaculum intestinale Human - fecal Yeast FattyAcid Casitone Media 233 Oscillibacter ruminantium Human - fecal YeastFatty Acid Casitone Media 234 Oscillibacter ruminantium Human - fecalYeast Fatty Acid Casitone Media 235 Oscillibacter ruminantium Human -fecal Yeast Fatty Acid Casitone Media 236 Oscillibacter ruminantiumHuman - fecal Yeast Fatty Acid Casitone Media 237 Parabacteroidesdistasonis Human - fecal Yeast Fatty Acid Casitone Media 238Parabacteroides merdae Human - fecal Yeast Fatty Acid Casitone Media 239Parabacteroides merdae Human - fecal Yeast Fatty Acid Casitone Media 240Parabacteroides merdae Human - fecal Yeast Fatty Acid Casitone Media 241Parabacteroides merdae Human - fecal Yeast Fatty Acid Casitone Media 242Parabacteroides merdae Human - fecal Yeast Fatty Acid Casitone Media 243Propionibacterium acnes Mus musculus - fecal Mucin Minimal Media 244Roseburia inulinivorans Human - fecal Yeast Fatty Acid Casitone Media245 Roseburia inulinivorans Human - fecal Yeast Fatty Acid CasitoneMedia 246 Roseburia inulinivorans Human - fecal Yeast Fatty AcidCasitone Media 247 Ruminococcus bromii Human - fecal Yeast Fatty AcidCasitone Media 248 Shigella flexneri Human - fecal Yeast Fatty AcidCasitone Media 249 Staphylococcus epidermidis Human - fecal Yeast FattyAcid Casitone Media 250 Staphylococcus warneri Human - fecal Yeast FattyAcid Casitone Media 251 Sutterella wadsworthensis Mus musculus - fecalMucin Minimal Media

The present disclosure provides compositions and methods that cancomprise at least one bacterial strain of Table 1. The presentdisclosure provides compositions and methods that can comprise at leasttwo bacterial strains of Table 1. The present disclosure providescompositions and methods that can comprise at least three bacterialstrains of Table 1. The present disclosure provides compositions andmethods that can comprise at least about 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or least about 30bacterial strains of Table 1.

The present disclosure provides compositions and methods that cancomprise one or more of the bacterial strains 1-25 described in Table 2below, or any combination of strains 1-25. The isolated strains of Table2 have been numbered as strains 1-25. The isolate number of such strainscorresponds to the isolate numbers shown in Table 1.

TABLE 2 Examples of Strains Used in Therapeutic Consortia Isolate StrainNumber Number Bacterial Strain 1 1 A. muciniphila ST7 2 2 A. muciniphilaST6 4 3 A. muciniphila ST5 5 4 A. muciniphila ST1 (Am1) 11 5 A.muciniphila ST15 (Am5) 18 6 A. muciniphila ST22 (Am6) 28 7 A.muciniphila ST38 (Am4) 31 8 A. muciniphila ST43 (Am2) 33 9 A.muciniphila ST46 (Am3) 39 10 A. muciniphila ST58 (Am7) 111 11 B.adolescentis ST15 (Bs10) 113 12 B. longum ST27 (Bl9) 126 13 B. productaST4 (Bp8) 147 14 C. comes ST3 (Cc11) 184 15 F. prausnitzii ST23 (Fp13)188 16 F. prausnitzii ST27 (Fp14) 192 17 F. prausnitzii ST38 (Fp12) 19518 F. prausnitzii ST58 (Fp15) 199 19 F. prausnitzii ST74 (Fp16) 211 20L. symbiosum ST40 (Bp7) 214 21 L. crispatus ST100 (Lj20) 221 22 L.jensenii ST10 (Lc18) 223 23 L. johnsonii ST8 (Lg19) 225 24 L. johnsoniiST74 (Lj 17) 247 25 R. bromii ST42 (Rb21)

The present disclosure provides compositions and methods that cancomprise of various microbial compositions. Such compositions cancomprise any of the bacterial strains 1-25 (e.g., those listed in Table2), or any combination of bacterial strains 1-25. Thus, a microbialcomposition can comprise at least one bacterial strain of Table 2. Amicrobial composition can comprise at least about two bacterial strainsof Table 2. A microbial composition can comprise at least about threebacterial strains of Table 2. A microbial composition can comprise atleast about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or least about 25 bacterial strains of Table 2.

A microbial composition as described herein can comprise one or moreAkkermansia muciniphila strains, one or more Faecalibacteriumprausnitzii strains, and/or one or more Lactobacillus crispatus strains.The compositions and methods described herein can comprise a microbialcomposition (e.g., a therapeutic microbial composition) that cancomprise at least the Akkermansia muciniphila strain A. muciniphila ST7(e.g., strain 1). The compositions and methods described herein cancomprise a microbial composition (e.g., a therapeutic microbialcomposition) that can comprise at least the Faecalibacterium prausnitziistrain F. prausnitzii ST38 (e.g., strain 17). The compositions andmethods described herein can comprise a microbial composition (e.g., atherapeutic microbial composition) that can comprise at least theLactobacillus crispatus strain L. crispatus ST100 (e.g., strain 21).

A microbial composition of the present disclosure can comprise any oneor more of the bacterial strains A. muciniphila ST7, F. prausnitziiST38, or L. crispatus ST100, or any combination thereof. A microbialcomposition described herein can comprise at least the bacterial strainsA. muciniphila ST7, F. prausnitzii ST38, and L. crispatus ST100.

A microbial composition as described herein can achieve certainbiological effects (e.g., therapeutic effects) in a synergistic manner.A synergistic bacterial consortium can comprise any one or more of thebacterial strains A. muciniphila ST7, F. prausnitzii ST38, or L.crispatus ST100, or any combination thereof. Such synergistic effects ofa microbial composition can include metabolic interaction between cellsof different bacterial strains (e.g., cells of a first strain produce ametabolite that can be used metabolically by cells of a second strain).Such synergistic effects of a microbial composition can includetherapeutic effects when administered to a subject (e.g., a rodent or ahuman). The metabolic activity of a bacterial composition describedherein can have a synergistic therapeutic effect, such asanti-inflammatory effects, when administered to a subject in needthereof.

The microbial compositions described herein that can comprise any one ormore of the bacterial strains A. muciniphila ST7, F. prausnitzii ST38,or L. crispatus ST100, or any combination thereof can be synergisticbacterial consortia. Synergistic effects of such a consortium caninclude increased therapeutic efficacy in a subject and beneficiallong-term effects for said subject. A microbial composition of thepresent disclosure can comprise one or more bacterial species and/orstrains that can produce one or more therapeutically effectivecompounds. Such compounds can have anti-inflammatory effects.

The production of such compounds can contribute to beneficial effects ofa species or strain, or that of a microbial composition that comprisessuch species or strain(s). The therapeutic compositions and consortia ofthe present disclosure can produce various compounds and/or metabolitesthat can have therapeutic and/r preventative properties whenadministered to a subject in need thereof. Such compounds andmetabolites can include fatty acids (e.g., SCFAs such as acetic acid orbutyric acid), lipids, phospholipids (e.g., phosphatidylcholines and/orderivatives thereof), and/or other metabolites with beneficial (e.g.,health-promoting) properties (e.g., when administered to a subject).

The compositions and methods described herein can comprise one or morebacterial cells of one or more bacterial species or strains that produceshort-chain fatty acids (SCFAs). The compositions and methods describedherein can comprise one or more bacterial cell of one or more bacterialspecies or strains that produce phospholipids and/or derivativesthereof. Such phospholipid and/or derivative thereof can be aphosphatidylcholine and/or a derivative thereof. The present disclosureprovides one or more species or strains of Akkermansia sp. that canproduce one or more phospholipids and/or derivatives thereof. TheAkkermansia strain that produces one or more phospholipids can be A.muciniphila ST7. At least one of the phospholipids that can be producedby Akkermansia sp. (e.g., A. muciniphila ST7) can be aphosphatidylcholine, a phopshatidylcholine-like compound, and/or achemical derivative thereof. Such compounds can be, or can havestructural similarity to compounds 1-3 shown in FIG. 13 . Such compoundscan be, or can have structural similarity to any one ofphosphatidylcholine-derived compounds including[3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-hydroxypropyl]tetradecanoate,[3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-hydroxypropyl]pentadecanoate, or[3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-hydroxypropyl] hexadecanoate,or chemical derivatives thereof.

Therapeutic Microbial Consortia

Generally, a therapeutic microbial consortium (or population) of thepresent disclosure can comprise one or more genera, species, strains,and/or strain variants belonging to the phyla Verrucomicrobia,Firmicutes, Proteobacteria, Actinobacteria, and/or Bacteroidetes, or anycombination thereof. A therapeutic microbial consortium can comprisebacteria belonging to one or more of the genera Faecalibacterium sp.,Akkermansia sp., Lactobacillus sp., or any combination thereof.

A therapeutic microbial consortium can comprise less than about 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 differentspecies of bacteria. A therapeutic microbial consortium can compriseless than about 20 different species of bacteria A therapeutic microbialconsortium can comprise less than 20 different species of bacteria. Atherapeutic microbial consortium can comprise less than about 15different species of bacteria. A therapeutic microbial consortium cancomprise less than 15 different species of bacteria. A therapeuticmicrobial consortium can comprise less than about 10 different speciesof bacteria. A therapeutic microbial consortium can comprise less than10 different species of bacteria. A therapeutic microbial consortium cancomprise less than about 9 different species of bacteria. A therapeuticmicrobial consortium can comprise less than 9 different species ofbacteria. A therapeutic microbial consortium can comprise less thanabout 8 different species of bacteria. A therapeutic microbialconsortium can comprise less than 8 different species of bacteria. Atherapeutic microbial consortium can comprise less than about 7different species of bacteria. A therapeutic microbial consortium cancomprise less than 7 different species of bacteria. A therapeuticmicrobial consortium can comprise than about 6 different species ofbacteria. A therapeutic microbial consortium can comprise than 6different species of bacteria. A therapeutic microbial consortium cancomprise less than about 5 different species of bacteria. A therapeuticmicrobial consortium can comprise less than 5 different species ofbacteria. A therapeutic microbial consortium can comprise less thanabout 4 different species of bacteria. A therapeutic microbialconsortium can comprise less than 4 different species of bacteria. Atherapeutic microbial consortium can comprise less than about 3different species of bacteria. A therapeutic microbial consortium cancomprise less than 3 different species of bacteria. A therapeuticmicrobial consortium can comprise less than about 2 different species ofbacteria. A therapeutic microbial consortium can comprise less than 2different species of bacteria.

A therapeutic microbial consortium (i.e., therapeutic consortium, alsoabbreviated herein as “TC”) of the present disclosure can providetherapeutically effective amounts of said bacterial consortium to asubject in need thereof. The therapeutic consortium can comprise,consist essentially of, or consist of any of the bacterial specieslisted in Table 1, or any combination thereof. The present disclosureprovides therapeutic microbial compositions that can comprise at leastone Akkermansia species. A therapeutic microbial composition cancomprise at least one Faecalibacterium species. A therapeutic microbialcomposition can comprise at least one Lactobacillus species. Atherapeutic microbial composition can comprise at least one Akkermansiaspecies and at least one Faecalibacterium species. A therapeuticmicrobial composition can comprise at least one Akkermansia species andat least one Lactobacillus species (Lactobacillus sp.). A therapeuticmicrobial composition can comprise at least one Faecalibacterium species(Faecalibacterium sp.) and at least one Lactobacillus species. Thepresent disclosure provides therapeutic microbial compositions that cancomprise at least one Akkermansia species, at least one Faecalibacteriumspecies and at least one Lactobacillus species. A therapeutic microbialcomposition can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or least 25 bacterialspecies of Table 1.

Such species can be Lactobacillus crispatus, Faecalibacteriumprausnitzii, Akkermansia muciniphila, and/or a Bifidobacterium species.A therapeutic microbial consortium of the present disclosure cancomprise the bacterial species Lactobacillus crispatus, Faecalibacteriumprausnitzii, Akkermansia muciniphila, and/or a Bifidobacterium species.Such consortia can be administered to a subject in need to treatdiseases such as dysbiosis, asthma, inflammation, and/or allergy.

A therapeutic bacterial consortium of the present disclosure cancomprise any one or more of the bacterial strains listed in Table 1and/or Table 2, or any combination thereof. The present disclosureprovides therapeutic microbial compositions and methods that cancomprise at least one bacterial strain of Table 1. The presentdisclosure provides therapeutic microbial compositions and methods thatcan comprise at least two bacterial strains of Table 1. The presentdisclosure provides therapeutic microbial compositions and methods thatcan comprise at least three bacterial strains of Table 1. The presentdisclosure provides therapeutic microbial compositions and methods thatcan comprise at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or least about 30 bacterialstrain of Table 1. The present disclosure provides therapeutic microbialcompositions and methods that can comprise of various microbialcompositions. Such therapeutic microbial compositions can comprise anyof the bacterial strains 1-25 (e.g., those listed in Table 2), or anycombination of bacterial strains 1-25. Thus, a therapeutic microbialcomposition can comprise at least one bacterial strain of Table 2. Atherapeutic microbial composition can comprise at least about twobacterial strains of Table 2. A therapeutic microbial composition cancomprise at least about three bacterial strains of Table 2. Atherapeutic microbial composition can comprise at least about 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, orleast about 25 bacterial strains of Table 2.

A therapeutic bacterial consortium of the present disclosure cancomprise at least one A. muciniphila strain. Such at least one straincan be A. muciniphila ST7. A therapeutic bacterial consortium of thepresent disclosure can comprise at least one F. prausnitzii strain. Suchat least one strain can be F. prausnitzii ST38. A therapeutic bacterialconsortium of the present disclosure can comprise at least one L.crispatus strain. Such strain can be L. crispatus ST100. A therapeuticbacterial consortium of the present disclosure can comprise at least oneA. muciniphila strain and at least one F. prausnitzii strain. The atleast one A. muciniphila strain can be A. muciniphila ST7, and the atleast one F. prausnitzii strain can be F. prausnitzii ST38. Atherapeutic bacterial consortium of the present disclosure can compriseat least one A. muciniphila strain and at least one L. crispatus strain.The at least one A. muciniphila strain can be A. muciniphila ST7, andthe at least one L. crispatus strain can be L. crispatus ST100. Atherapeutic bacterial consortium of the present disclosure can compriseat least one F. prausnitzii strain and at least one L. crispatus strain.The at least one F. prausnitzii strain can be F. prausnitzii ST38, andthe at least one L. crispatus strain can be L. crispatus ST100. Atherapeutic bacterial consortium of the present disclosure can compriseat least one A. muciniphila strain, at least one F. prausnitzii strainand at least one L. crispatus strain. The at least one A. muciniphilastrain can be A. muciniphila ST7, the at least one F. prausnitzii straincan be F. prausnitzii ST38, and the at least one L. crispatus strain canbe L. crispatus ST100. A therapeutic bacterial consortium of the presentdisclosure can comprise A. muciniphila ST7, F. prausnitzii ST38, or L.crispatus ST100. Any one of these strains, or any combination thereof,can be combined with any of the strains 1-25 of Table 1 and/or any ofthe strains of Table 2.

A therapeutic bacterial consortium of the present disclosure cancomprise one or more variants (e.g., genetic variants) of a certainstrain. For example, therapeutic consortium can comprise any one or moreof the A. muciniphila ST7 (or AM ST7) variants AM-ST7_1, AM-ST7_2, andAM-ST7_3 (see e.g., FIG. 4 -FIG. 6 ).

A therapeutic consortium or composition as disclosed herein can have oneor more beneficial and/or therapeutic properties. Such therapeuticproperties can include anti-inflammatory properties, anti-allergic,anti-infective properties, or anti-cancer properties. Such propertiescan include reduction of one or more pro-inflammatory markers and/ormetabolites. These can include certain immunoglobulins (e.g., IgE),histamines, pro-inflammatory chemokines or cytokines (e.g., IL-4, IL-13,etc.), certain T helper cells (e.g., T_(H)2 or T_(H)17 cells), orcertain immune cells such as eosinophils, neutrophils, mast cells, orbasophils. The anti-inflammatory compound, marker, and/or metabolite canbe a cytokine, a microbial lipid, a microbial carbohydrate, a microbialfatty acid, or a microbial amino acid. The anti-inflammatory compoundcan be IL-17. The pro-inflammatory compound can be a cytokine, amicrobial lipid, a microbial carbohydrate, or a microbial amino acid.The pro-inflammatory compound can be IL-4, IL-10, IL-8, IL-13, TNF-a, orMUC5B, or any combination thereof. The microbial lipid can be aphospholipid. The phospholipid can be a phosphatidylcholine or aphosphatidylcholine-like compound (e.g., those listed in FIG. 13 , orderivatives thereof, or those having the chemical names of any one ofphosphatidylcholine-derived compounds including[3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-hydroxypropyl]tetradecanoate,[3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-hydroxypropyl]pentadecanoate, or[3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-hydroxypropyl] hexadecanoate,strcuturally similar derivatives thereof, or thosephosphatidylcholine-derived compounds that are shown in FIG. 13 ).

A microbial composition of the present disclosure can comprise one ormore bacterial species and/or strains that can produce one or moretherapeutically effective compounds. Such compounds can be smallmolecules (e.g., fatty acids, lipids, etc.), peptides, polypeptide,and/or nucleic acids. Such compounds can have beneficial effects in asubject (e.g., a rodent or a human) when administered to said subject(e.g., in an oral formulation). Such beneficial effects can includeanti-inflammatory effects. Such anti-inflammatory effects can besystemic, i.e., anti-inflammatory effects can be elicited in variousparts of the body of an organism (e.g., a mammal such as a human). Thetherapeutic consortia described herein can comprise one or morebacterial cells of one or more bacterial genera, species or strains thatcan produce small molecule metabolites with beneficial (e.g.,therapeutic) properties. The beneficial small molecules can includefatty acids such as produce short-chain fatty acids (SCFAs) and/orlipids such as phospholipids. In particular, the compositions andmethods described herein can comprise one or more bacterial cells of oneor more bacterial genera, species or strains that produce phospholipidsand/or derivatives thereof. Such phospholipid and/or derivative thereofcan be a phosphatidylcholine, a phosphatidylcholine-like compound and/ora phosphatidylcholine derivative (e.g., a chemically and/or structurallysimilar molecule to a phosphatidylcholine). The present disclosureprovides one or more species and/or strains of Akkermansia sp. that canproduce one or more phospholipids and/or derivatives thereof. TheAkkermansia strain that produces one or more phospholipids can be A.muciniphila ST7. At least one of the phospholipids that can be producedby A. muciniphila ST7 can be a phosphatidylcholine and/or a derivativethereof. Such compounds can have structural similarity with compounds1-3 shown in FIG. 14 . The production of one or morephosphatidylcholine(s) or derivatives thereof by Akkermansia sp. (e.g.,A. muciniphila ST7) can have an anti-inflammatory effect in a subject(e.g., a human).

A therapeutic consortium of the present disclosure, once administered toa subject, can affect the metabolism of one or more compounds in thesubject (e.g., a human or non-human animal). Affecting the metabolism ofone or more compounds in the subject can be beneficial for said subject,e.g., in terms of having preventative and/or therapeutic properties(e.g., anti-inflammatory properties). A therapeutic consortium of thepresent disclosure can affect and/or alter the metabolism of fatty acidsand/or lipids in a subject. A therapeutic consortium can affect and/oralter the metabolism of phospholipids and unsaturated orpoly-unsaturated fatty acids. Such fatty acids can include omega-3-and/or omega-6 fatty acids. The metabolic effects that a therapeuticconsortium can elicit include the metabolism of linoleic acid (e.g.,alpha-linolenic acid), arachidonic acid, and other fatty acids. Themetabolism of such fatty acids can be affected in a way that results ina reduction of inflammation in a subject. A therapeutic consortium canaffect metabolic pathways used to metabolize certain compounds such asfatty acids, lipids, etc. Affecting such metabolic pathways can includeat least a partial of full inhibition of certain metabolic pathways,such as pathways that result in the production of pro-inflammatorycompounds or metabolites and/or compounds or metabolites that areassociated with inflammation. A therapeutic consortium can affect and/oralter a metabolic pathway directly and/or indirectly. For example, atherapeutic consortium of the present disclosure can directly and/orindirectly increase the anti-inflammatory effects of alpha-linolenicacid by reducing the amount of alpha-linolenic acid metabolite(s) thatmay mediate inflammation and thus counteract the anti-inflammatoryeffects of that lipid. Thus, a therapeutic consortium can beneficiallyaffect and/or alter a metabolism in a subject. This can result inreducing the incidence of inflammation (e.g., chronic and/or allergicinflammation), a metabolic disease or disorder, an allergy, a dysbiosis,a cancer, or any combination thereof

A therapeutic consortium of the present disclosure can producebeneficial fatty acids, SCFAs, lipids, and/or phospholipids (e.g.,phosphatidylcholine or a derivative thereof). The amount of any of suchcompounds produced by a therapeutic consortium can be at least about1.1, 1.2, 1.3., 1.4, 1.5., 1.6, 1.7, 1.8., 1.9., 2, 2.2, 2.5, 2.8, 3,3.5, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 times the amount produced by aconsortium that does not contain all the bacterial genera, species,and/or strains of the therapeutic consortium.

A therapeutic consortium of the present disclosure can comprise A.muciniphila sp. Such a therapeutic consortium can produce one or morephosphatidylcholine(s) and/or derivatives thereof. The consortiumcomprising A. muciniphila sp. can produce at least about 1.1, 1.2, 1.3.,1.4, 1.5., 1.6, 1.7, 1.8., 1.9., 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 6, 7,8, 9, 10, 20, 50, or 100 times the amount of a phosphatidylcholine or aderivative thereof than a consortium that does not contain A.muciniphila sp.

A therapeutically effective microbial consortium as provided herein canrefer to a therapeutically effective composition including a bacterialpopulation that comprises, consists essentially of, or consists of any1, 2, 3, 4, 5, 6, 7, or 8 of Lactobacillus species and/or strains,Faecalibacterium species and/or strains, and/or Akkermansia speciesand/or strains, or any combination thereof. A microbial composition asdescribed herein can comprise one or more Bifidobacterium species and/ostrains. The composition can include less than about 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 different species ofbacteria.

A composition provided herein can be administered orally and can includelive microorganisms at a concentration of at least about 10³, 10⁴, 10⁵,10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or greater units per gram (cfu/g). Thecomposition provided herein can be administered orally and includes livemicroorganisms from about 10⁷ to about 10¹¹ cfu/g. The composition caninclude from about 10⁴ to about 10¹⁵ cfu/g of a bacterial species orstrain. The composition can include from about 10⁵ to about 10¹⁵ cfu/gof a bacterial species or strain. The composition can include from about10⁶ to about 10¹⁵ cfu/g of a bacterial species or strain. Thecomposition can include from about 10⁷ to about 10¹⁵ cfu/g of abacterial species or strain. The composition can include from about 10⁸to about 10¹⁵ cfu/g of a bacterial species or strain. The compositioncan include from about 10⁹ to about 10¹⁵ cfu/g. The composition caninclude from about 10¹⁰ to about 10¹⁵ cfu/g of a bacterial species orstrain. The composition can include from about 10¹¹ to about 10¹⁵ cfu/gof a bacterial species or strain. The composition can include from about10¹² to about 10¹⁵ cfu/g of a bacterial species or strain. Thecomposition can include from about 10¹³ to about 10¹⁵ cfu/g of abacterial species or strain. The composition can include from about 10¹⁴to about 10¹⁵ cfu/g of a bacterial species or strain. The compositioncan include from about 10³ to about 10¹⁵ cfu/g of a bacterial species orstrain.

A bacterial composition as provided herein can be administered orally(e.g., as a solid dosage from or as a liquid such as a solution orsuspension) and can include at least about 10⁷ to about 10¹¹ cfu perbacterial species or strain. A composition provided herein can beadministered orally and can include at least about 10⁷ to about 10¹³ cfuper bacterial specie or strain. A composition provided herein can beadministered orally and can include at least about 10⁵ to about 10¹⁰ cfuper bacterial species or strain. A composition provided herein can beadministered orally and can include at least about 10⁴ to about 10⁹ cfuper bacterial species or strain. A composition provided herein can beadministered orally and can include at least about 10⁵ to about 10⁸ cfuper bacterial species or strain.

Pharmaceutical Compositions

A microbial composition (e.g., a therapeutic bacterial consortium) ofthe present disclosure can be used and/or administered as apharmaceutical composition. Such a pharmaceutical composition cancomprise one or more pharmaceutically acceptable excipients and/orcarriers.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” can refer to a substance that aids the administration of anactive agent (e.g., a microbial composition) to and absorption by asubject and can be included in the compositions of the presentdisclosure without causing a significant adverse toxicological effect ona subject (e.g., a rodent or a human). Non-limiting examples ofpharmaceutically acceptable excipients can include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and/or mixed withauxiliary agents such as lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the present disclosure. Anexcipient can consume oxygen, e.g., reducing the oxygen content of orwithin a composition or pharmaceutical composition. Reducing the oxygencontent may increase the viability of the bacterial cells in thatcomposition, in cases where e.g., the bacteria are anaerobic bacteria.

The microbial compositions provided herein can be administered orally,gastrointestinally, or rectally. Administration can be in the form of asingle bolus dose, or may be, for example, by a continuous perfusionpump. The microbial consortium provided herein can be combined with oneor more excipients, for example, a disintegrant, a filler, a glidant, ora preservative. The microbial consortium provided herein can form partof a capsule. Suitable capsules include both hard shell capsules orsoft-shelled capsules. Any lipid-based or polymer-based colloid can beused to form the capsule. Exemplary polymers useful for colloidpreparations include gelatin, plant polysaccharides or their derivativessuch as carrageenans and modified forms of starch and cellulose, e.g.,hypromellose. Optionally, other ingredients can be added to the gellingagent solution, for example plasticizers such as glycerin and/orsorbitol to decrease the capsule's hardness, coloring agents,preservatives, disintegrants, lubricants and surface treatment.

The microbial compositions of the present disclosure can be formulatedin a unit dosage form, each dosage containing, for example, from about0.005 mg to about 2000 mg of a defined microbial consortium havingminimal urease activity per dose. The term “unit dosage forms” can referto physically discrete units suitable as unitary dosages for humansubjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient. For preparing solid compositions such as tablets, theprincipal active ingredient can be mixed with a pharmaceutical excipientto form a solid preformulation composition containing a homogeneousmixture of a compound of the present disclosure. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation canthen be subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.005 mg to about 1000 mg of the microbialcomposition provided herein.

The microbial compositions of the present disclosure can be formulatedin a unit dosage form, each dosage containing from about 0.1 mg to about50 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 20mg from about 0.1 mg to about 10 mg, from about 0.2 mg to about 20 mg,from about 0.3 mg to about 15 mg, from about 0.4 mg to about 10 mg, fromabout 0.5 mg to about 1 mg; from about 0.5 mg to about 100 mg, fromabout 0.5 mg to about 50 mg, from about 0.5 mg to about 30 mg, fromabout 0.5 mg to about 20 mg, from about 0.5 mg to about 10 mg, fromabout 0.5 mg to about 5 mg; from about 1 mg from to about 50 mg, fromabout 1 mg to about 30 mg, from about 1 mg to about 20 mg, from about 1mg to about 10 mg, from about 1 mg to about 5 mg; from about 5 mg toabout 50 mg, from about 5 mg to about 20 mg, from about 5 mg to about 10mg; from about 10 mg to about 100 mg, from about 20 mg to about 200 mg,from about 30 mg to about 150 mg, from about 40 mg to about 100 mg, fromabout 50 mg to about 100 mg of a Lactobacillus sp. (e.g., L. crispatusST100), Faecalibacterium sp. (e.g., F. prausnitzii ST38), and/or anAkkermansia sp. (e.g., A. muciniphila ST7), either individually orcombined.

Tablets or pills that can be used in combination with the microbialcompositions of the present disclosure can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. A tablet or pill can comprise an inner dosage and an outerdosage component, the latter being in the form of an envelope over theformer. The two components can be separated by an enteric layer whichserves to resist disintegration in the stomach and permit the innercomponent to pass intact into the duodenum or to be delayed in release.A variety of materials can be used for such enteric layers or coatings,such materials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate. The liquid forms in which the compositions of thepresent disclosure can be incorporated for administration orally or byinjection include aqueous solutions, suitably flavored syrups, aqueousor oil suspensions, and flavored emulsions with edible oils such ascottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Methods of Treatment

The therapeutic microbial compositions and methods described herein canbe used for the prevention and/or treatment of a disease or condition ina subject (e.g., a rodent or a human).

Therapeutic treatment using the herein described microbial compositions(e.g., those comprising one or more of A. muciniphila ST7, F.prausnitzii ST38, or L. crispatus ST100, or a combination thereof) caninclude administering to a subject a therapeutically effective amount ofsaid microbial composition (e.g., as a pharmaceutical composition)before and/or after diagnosis or development of a disease, condition, ordisorder. Thus, a method of treating a disease (e.g., an inflammatorydisease, an infection, and/or a dysbiosis) in a subject in need thereofcan be provided by utilizing the herein described compositions andmethods.

The one or more bacteria of a therapeutic composition of the presentdisclosure can be delivered in a lyophilized form or a suspended (e.g.,liquid) from. The dosage that may be administered to a subject in needthereof can depend on the route of administration, the nature of theformulation, the nature of the subject's condition, e.g., immaturity ofthe immune system or a gastrointestinal disorder, the subject's size,weight, surface area, age, and sex, and/or other drugs beingadministered, and the judgment of the attending clinicians. Suitabledosages can be in the range of 0.01-1,000 mg/kg. Some typical doseranges can be from about 1 μg/kg to about 1 g/kg of body weight per day.The dose range can be from about 0.01 mg/kg to about 100 mg/kg of bodyweight per day. The dose can be, for example, 1 mg/kg, 2 mg/kg, 5 mg kg,10 mg/kg, 20 mg/kg, 50 mg/kg or 100 mg/kg. Alternatively or in addition,the dosage can be expressed as cfu or as cfu/g of dry weight. Thus, thedosage may vary, but can range from the equivalent of about 10² to about10¹² cfu/g, e.g., 1×10² cfu/g, 5×10² cfu/g, 1×10³ cfu/g, 5×10³ cfu/g,1×10⁴ cfu/g, 5×10⁴ cfu/g, 1×10⁵ cfu/g, 5×10⁵ cfu/g, 1×10⁶cfu/g, 5×10⁶cfu/g, 1×10⁷ cfu/g, 5×10⁷ cfu/g, 1×10⁸ cfu/g, 5×10⁸ cfu/g, 1×10⁹ cfu/g,5×10⁹ cfu/g, 1×10¹⁰ cfu/g 5×10¹° cfu/g, 1×10¹¹ cfu/g, 5×10¹¹ cfu/g, or1×10¹² cfu/g, or more of dry weight of any one of the administeredbacteria (individually) or of the total population of bacteria. Thedosage can range from about 10² to about 10¹² cfu, e.g., 1×10² cfu,5×10² cfu, 1 ×10³ cfu, 5×10³ cfu, 1×10⁴ cfu, 5×10⁴ cfu, 1×10⁵ cfu, 5×10⁵cfu, 1×10⁶ cfu, 5×10⁶ cfu, 1×10⁷ cfu, 5×10⁷ cfu, 1×10⁸ cfu, 5×10⁸ cfu,1×10⁹ cfu, 5×10⁹ cfu, 1×10¹° cfu, 5×10¹⁰ cfu, 1×10¹¹ cfu, 5×10¹¹ cfu, or1×10¹²cfu of any one of the administered bacteria (individually) or ofthe total population of bacteria (e.g., A. muciniphila ST7, F.prausnitzii ST38, or L. crispatus ST100, or any combination thereof).

The methods and compositions of the present disclosure can beadministered to a subject in need thereof. The subject may suffer from adisease or conditions. The subject can be a neonate, an infant, atoddler, a child, a teenager, an adult, or a subject of any age. Theneonate can be less than about 3 days old. The neonate can be less thanabout 1 week old. The neonate can be less than about 2 weeks old. Theneonate can be less than about 3 weeks old. The neonate can be less thanabout 4 weeks old. The infant can be less than about 8 weeks old. Theinfant can be at least about 2 months old. The infant can be at least 6about months old. The infant can be at least about 12 about months old.

The methods and compositions of the present disclosure can beadministered to a subject of varying ages and maturity. The microbialcompositions can be used to treat a subject that can be between about 2and about 18 years old, or can be at least about 18 years old. Thesubject can be between 2 and 18 years old, or is at least 18 years old.The subject can be between about 2 and about 18 years old, or is atleast about 18 (e.g., 19, 20, 25, 30, 40, 50, 60, 70, 80, 90) years old.The subject can be between about 2 and about 18 years old, or is about19 years old. The subject can be between about 2 and about 18 years old,or is 19 years old. The subject can be between about 2 and about 18years old, or is about 20 years old. The subject can be between about 2and about 18 years old, or can be 20 years old. The subject can bebetween about 2 and about 18 years old, or can be about 25 years old.The subject can be between about 2 and about 18 years old, or can be 25years old. The subject can be between about 2 and about 18 years old, orcan be about 30 years old, or older. The subject can be between about 2and about 18 years old, or can be 30 years old. The subject can bebetween about 2 and about 18 years old, or can be about 40 years old.The subject can be between about 2 and about 18 years old, or can be 40years old. The subject can be less than about 50 years old. The subjectcan be less than about 60 years old. The subject can be at least about60 years old. The subject can be at least about 80 years old.

The compositions and methods described herein can be used to preventand/or treat various disorders, conditions, or disease, and can beadministered for various periods of time. A treatment period may varybetween subjects and individuals and can depend on various factors asdescribed herein, e.g., disease state, age, etc. A subject can betreated for at least about one day to at least about one week. Thesubject can be treated for at least about a week to at least about onemonth. The subject can be treated for at least about one month to atleast about one year. The subject can be treated for at least about twomonths. The subject can be treated for at least about six months. Thesubject can be treated for at least twelve months. The subject can betreated for at least two years. The subject can be treated onconsecutive days, consecutive weeks, and/or consecutive months.

The methods and compositions of the present disclosure can be used toprevent and/or treat various disorders, conditions, or diseases. Suchdisorders, conditions, or diseases can be caused by dysbiosis. Thedysbiosis can be dysbiosis of a gut microbial composition. Dysbiosis canbe described as a microbial imbalance within the human gut. Microbialimbalance can include the overgrowth of potentially pathogenic bacterialeading to changes in functional and metabolic compositions of the gutflora.

The disease to be prevented and/or treated can be an inflammatorydisease. The inflammatory disease can be an allergy, atopy, asthma, anautoimmune disease, an autoinflammatory disease, a hypersensitivity,pediatric allergic asthma, allergic asthma, inflammatory bowel disease,Celiac disease, Crohn's disease, colitis, ulcerative colitis,collagenous colitis, lymphocytic colitis, diverticulitis, irritablebowel syndrome, short bowel syndrome, stagnant loop syndrome, chronicpersistent diarrhea, intractable diarrhea of infancy, Traveler'sdiarrhea, immunoproliferative small intestinal disease, chronicprostatitis, postenteritis syndrome, tropical sprue, Whipple's disease,Wolman disease, arthritis, rheumatoid arthritis, Behcet's disease,uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury,psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis,systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onsetdiabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto'sencephalitis, Hashimoto's thyroiditis, ankylosing spondylitis,psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis,auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis,Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris,pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplantrejection, interstitial cystitis, atherosclerosis, or atopic dermatitis.

The inflammatory disease to be treated and/or prevented using the hereindescribed microbial consortia can be a pediatric condition such aspediatric allergic asthma or inflammatory bowel disease. The pediatricsubject may suffer from constipation, diarrhea, bloating, urgency,and/or abdominal pain.

Therapeutically effective bacterial consortia as described herein cancomprise, consist essentially of, or consist of the bacterial speciesLactobacillus johnsonii or Lactobacillus crispatus, Faecalibacteriumprausnitzii, Akkermansia muciniphila, or a Bifidobacterium species,strains thereof, and/or any combination thereof. Such microbialcompositions can be used to treat pediatric indications, includingpediatric dysbiosis and pediatric inflammatory diseases. Such abacterial consortium can comprise any one or more of the bacterialstrains A. muciniphila ST7, F. prausnitzii ST38, or L. crispatus ST100,or any combination thereof. A microbial composition described herein cancomprise at least the bacterial strains A. muciniphila ST7, F.prausnitzii ST38, and L. crispatus ST100.

Containers, Kits, and Instructions

The present disclosure provides containers and kits that can be used incombination with the herein described microbial (e.g., bacterial)compositions (e.g., therapeutic microbial compositions). The presentdisclosure further provides instructions that can direct a user (e.g., ahuman user) to use the microbial compositions as well as the containersand kits that comprise such microbial compositions.

Containers of the present disclosure can be used to grow, store,transport, aliquot, and/or administer therapeutic microbialcompositions. Containers can be used to administer a therapeuticmicrobial composition to a subject (e.g., a human subject). Suchcontainers can, for example, provide conditions suitable for growth,transport, and/or storage (e.g., cooled or frozen storage) of microbialpopulations, e.g., those populations that comprise one or more anaerobicbacterial cells. In such cases, a container may be used to provide acertain oxygen content or concentration during growth, transport, and/orstorage of a therapeutic microbial composition in order to preserve theviability of the bacteria. Containers can further be used to providesuitable volumes, amounts, and schedules for administration a microbialcomposition to a subject (e.g., self-administration).

Kits of the present disclosure provide various components for using themicrobial compositions described herein. Such components can includecontainers, test samples, equipment for analyzing the composition, e.g.,its viability, pH of the storage medium, etc. Generally, kits allow foruser-friendly, accurate and reliable use of the therapeutic compositionsdescribed herein, including, but not limited to dosing, administration,storage, transport, etc.

The present disclosure provides instructions for using kits and/orcontainers in combination with the therapeutic compositions describedherein. Such instructions can be written instructions or oralinstructions, or a combination thereof. Such instructions can direct auser to use the therapeutic microbial compositions as well as thecontainers and kits that comprise such microbial compositions.

Procedures and Methods

General Sample Preparation. Samples such as biological samplescomprising one or more bacterial species can be collected and preparedusing various methodologies. The sample may be a fecal sample obtainedfrom a subject, such as a rodent or a human.

Fecal samples can be collected from subjects. Subsequent to delivery,these samples can be divided into aliquots (e.g., 0.5 g aliquots). Onetube from each sample can be frozen immediately at −80° C. One tube ofeach sample can be stored at +4° C. for several hours (e.g., one forabout 6 h and the other for about 24 h). Two additional tubes can beincubated at room temperature and collected at the same time points(e.g., 6 and 24 h). At the end point, samples can be rapidly frozen at−80° C. for subsequent fecal DNA extraction as described below.

Aliquots of (e.g., 0.5 g) of feces can be resuspended in 10 mL of SMbuffer and homogenized by vigorous vortexing for at least 5 min. Tubescan then be cooled on ice for 5 min prior to centrifugation at 5000 rpmfor 10 min at +4° C. Supernatants can be transferred to new tubes, andthe centrifugation step can be repeated one or more times. Supernatantscan be subsequently filtered twice through a 0.45-millipore PESsyringe-mounted membrane filters. NaCl and PEG-8000 powders can then beadded to filtrates to give a final concentration of 0.5 M and 10%(weight), respectively. Once the samples have been dissolved they can beincubated overnight at +4° C. The samples can be subsequentlycentrifuged at 5000 rpm for 20 min at +4° C. to collect precipitate.Pellets can be resuspended in 400 μl of SM buffer and extracted bygentle shaking with equal volume of chloroform. Emulsions can then becentrifuged at 2500 rpm for 5 min using a desktop centrifuge. Theaqueous phase (˜360 μl) can be aspirated into clean Eppendorf tubes andmixed with 40 μl of a solution of 10 mM CaCl₂ and 50 mM MgCl₂. Afteraddition of 8 U of TURBO DNase (Ambion/ThermoFisher Scientific) and 20 Uof RNase I (ThermoFisher Scientific) free DNA/RNA digestion can becarried out at 37° C. for 1 h before inactivating enzymes at 70° C. for10 min. Proteinase K (40 μg) and 20 μl of 10% SDS can then be added andto the tubes, and incubation was continued for 20 min at 56° C. Finally,viral particles can be lysed by addition of 100 μl of Phage Lysis Buffer(e.g., 4.5 M guanidinium isothiocyanate, 44 mM sodium citrate pH 7.0,0.88% sarkosyl, 0.72% 2-mercaptoethanol) and incubation at 65° C. for 10min. Ly-sates can then be extracted twice by gentle vortexing with equalvolume of Phenol/Chloroform/Isoamyl Alcohol 25: 24:1 (Fisher Scientific)followed by centrifugation at 8000 rpm for 5 min at room temperature.The resulting aqueous phase can be subjected to a final round ofpurification using DNeasy Blood & Tissue Kit (Qiagen), e.g., accordingto manufacturer's instruction with a final elution volume of 50 μl.

Sequencing. The microbial compositions of the present disclosure can beanalyzed and characterized using a variety of techniques. For example,iTag amplicon sequencing can be a barcoded 16S rRNA gene ampliconsequencing methodology that can be utilized to determine the relativeabundance of each taxon in a community, and/or to compare the taxonomicprofile between subject groups of interest. Amplicon sequencing analysiscan provide information on the overall microbial profile and its changesover time, both within and between treatment groups. The microbialcompositions of the present disclosure can also be characterized usingShotgun metagenomic sequencing, an effective environmental sequencingapproach that provides information about biodiversity and function of amicrobial community. The microbial compositions of the presentdisclosure may be analyzed and characterized using short-chain fattyacid (SCFA) analysis. Since SCFAs can be the main fermentation productsof gut microbiota and provide important insights into the relationshipbetween the gut microbiota and the host's physiology, their analysisoffers information about status, composition, and activity of the gutmicrobial composition. The health and disease status of a host as wellas the compositions and activities of the hosts' microbiome can beanalyzed and characterized using calprotectin analysis. Fecal sampleanalysis of calprotectin can provide information on inflammation in thegut. Moreover, fecal sample analysis of calprotectin can provideinformation on the composition of the microbial consortium of thesubject. Amplicon sequencing analysis can be performed for the taxonomicprofiling of prokaryotic communities (16S V4 region), eukaryoticcommunities (18S), fungal communities (ITS2), and archaeal communities(16S V4-V5 region), or any combination thereof.

The identification of isolate samples (e.g., strains) can be performedby PCR amplification of the full-length 16S rRNA gene. Such a method canuse one or more forward primer and/or one or more reverse primerfollowed by capillary sequencing. Full-length 16S rRNA gene sequencereads can be aligned in the Ribosomal Database Project (RDP), manuallycurated using nucleic acid analysis and sequencing programs (e.g., ARB,mother, etc.) to classify reads to operational taxonomic units (OTUs).The full-length 16S rRNA gene sequence of each species-level OTU canthen be compared to the RDP reference database to assign taxonomicdesignations to the genus and/or strain level followed by a BLASTnsearch to either a characterized or candidate novel species.

Sequencing results can be compared with databases, e.g., those of theHuman Microbiome Project (HMP), and can be carried out using 97%sequence similarity of the 16S rRNA gene sequence from the culturedbacteria to define a species when only partial 16S rRNA gene sequencesmay be available. Genomic DNA can be extracted from at least onerepresentative of each unique OTU using, for example, aphenol-chloroform-based DNA isolation procedure. DNA can then besequenced on an Illumina HiSeq platform generating read lengths of 100bp which can be assembled and annotated for further analysis. DNA can beextracted directly from each sample (e.g., a fecal sample) forwhole-community metagenomic and 16S rRNA gene amplicon sequencing usingthe MP Biomedical FastDNA SPIN Kit for soil. For comparisons with thecomplete community samples, non-confluent cultures can be scraped fromagar plates 72 h after inoculation with the initial fecal sample and DNAis extracted from this community using the same or similar DNA isolationprocess. 16S rRNA gene amplicon libraries are produced by PCRamplification of variable regions 1 and 2 of the 16S rRNA gene using theQ5 High-Fidelity Polymerase Kit supplied by New England Biolabs.

Shotgun Sequencing. Several microliters (e.g., 5-20 μL) of fecal nucleicacid sample regardless of concentration can be taken into reversetranscription reaction using SuperScript IV Reverse Transcriptase (RT)kit (Invitrogen/ThermoFisher Scientific), or similar equipment,according to the manufacturer's random hexamer primer protocol. Onemicroliter of reverse-transcribed nucleic acids is then amplified usingMDA technology with Illustra GenomiPhi V2 kit (GE Healthcare), andrepeated for each sample. Products from all three MDA reactions,together with the remainder of RT products, can be pooled together andsubjected to additional round of purification using DNeasy Blood &Tissue Kit. Amplified DNA can be quantified using Qubit dsDNA HS AssayKit (Invitrogen/ThermoFisher Scientific) and may be subjected to randomshotgun library preparation using Nextera XT DNA Library Preparation Kit(Illumina) and bead-based normalization following the standardmanufacturer's protocol. Ready-to-load libraries can be sequenced usinga proprietary modified protocol using e.g., 2×300 bp paired-endchemistry on an Illumina MiSeq platform (Illumina, San Diego, Calif.).

DNA Extraction and Library Preparation for Microbiota Profiling using16S rRNA Amplicon Sequencing. The QlAamp Fast DNA Stool Mini Kit(Qiagen, Hilden, Germany) can be used according to manufacturer'sguidelines to extract total fecal DNA from 200 mg aliquots of feces, butmay be modified to include a bead-beating step. The samples can beplaced in 2 mL screw-cap tubes containing 1 mL of InhibitEX Buffer and amixture of inert beads (ThistleScientific) of various diameters (one 3.5mm glass bead, 200 .tl of 1 mm zirconium beads, 200 .tl of 0.1 mmzirconium beads). Following 2×30s beating in FastPrep-24 instrument (MPBiomedicals) with an intermittent step of cooling on ice for 30 s, thesamples can be lysed by heating for 5 min at 95° C. Subsequently, thesamples are processed according to the standard Qiagen protocol.

Hypervariable regions V3 and V4 of bacterial 16S ribosomal RNA genes canbe amplified from 15 ng of total DNA template via PCR using PhusionHigh-Fidelity PCR Master Mix (ThermoFisher Scientific), appropriateprimers and overhang adapter sequences (sequence portions complementaryto bacterial 16S rRNA genes are underlined). The following PCR programcan be used: 98° C. 30 s, 25 cycles of 98° C. 10 s, 55° C. 15 s, 72° C.20 s, final extension 72° C. 5 min. Following purification usingAgencourt AMPure XP magnetic beads (Beckman-Coulter), the ampliconlibraries can undergo a second PCR reaction to attach dual IlluminaNextera indices using the Nextera XT index kit v2 (Illumina). Followingpurification (as described above), the dsDNA libraries can then bequantified using a Qubit dsDNA HS Assay Kit and pooled in equimolarconcentrations. Ready-to-load libraries can be sequenced using aproprietary modified protocol using 2×300 bp paired-end chemistry on anIllumina MiSeq platform (Illumina, San Diego, Calif.).

Analysis of 16S rRNA amplicon sequencing data. The quality of the rawreads can be visualized using FastQC (e.g., version v0.11.3). The readsare then imported into R (e.g., version v3.3.0) for data analysis withthe DADA2 package (e.g., version v1.03) Errors introduced during thesequencing process can be corrected to generate ribosomal sequencevariants (RSVs). These can be exported and further chimera filteredusing both the de novo and reference-based chimera filtering implementedin USEARCH v8.1.1861 with the ChimeraSlayer gold database v20110519. Theremaining RSVs are classified with mothur v1.38 against the RDP databaseversion 11.4, as well as classified with SPINGO to species level. OnlyRSVs with a domain classification of bacteria or archaea are kept forfurther analysis. A phylogenetic tree of the RSV sequences rooted on themidpoint is generated with FastTree.

Culturing of Microbial Strains and Species. The bacterial strains of thepresent disclosure may be obtained from a variety of sources. Thebacterial strains can be obtained from live sources or may be purchasedfrom commercially available providers, or a combination thereof. Themicroorganisms that the microbial consortia of the present disclosurecan be comprised of may be obtained from fecal samples (e.g., from ananimal such as a rodent or a human). The microorganisms (e.g., bacteria)that the microbial consortia of the present disclosure can be comprisedof may be obtained from human fecal samples.

Fecal samples are generally stored and handled under anaerobicconditions (see e.g., EXAMPLE 1 and EXAMPLE 2). Fresh fecal samples canbe stored and handled under anaerobic conditions within 1 h of passingto preserve the viability of anaerobic bacteria. All sample processingand culturing steps can be performed under anaerobic conditions. Culturemedia, PBS and all other materials that can be used for culturing can begenerally placed in the anaerobic cabinet at least 24 h before use toreduce to anaerobic conditions. Anaerobic culture methods can includethe use of Hungate culture tubes, sealed with butyl rubber septa (BellcoGlass), for example.

Homogenization of fecal samples can be performed using reduced PBS(e.g., using 0.1 g sample per ml PBS) and can be serially diluted andplated directly onto YCFA agar supplemented with approximately 0.002g/ml each of glucose, maltose and cellobiose in large (13.5 cm diameter)Petri dishes. In some instances, YCFA medium is used as growth mediumfor bacteria. The YCFA medium may generally contain (per 100 ml)Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g), cysteine (0.1g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g), (NH₄)₂SO₄ (0.09g), MgSO₄.7H₂O (0.009 g), CaCl₂ (0.009 g), resazurin (0.1 mg), hemin (1mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoic acid (3 μg), folicacid (5 μg), and pyridoxamine (15 μg). In addition, certain short-chainfatty acids (SCFA) are present including (final concentrations) acetate(33 mM); propionate (9 mM); isobutyrate, isovalerate, and valerate (1 mMeach). Cysteine can be added to the medium following boiling anddispensed into Hungate tubes while the tubes are flushed with CO₂. Afterautoclaving, filter-sterilized solutions of thiamine and riboflavin areadded to give final concentrations of 0.05 μg/ml of each. For someexperiments, the Casitone content can be decreased to 0.2%. Carbohydrateor other energy sources can be added as needed, and the final pH of themedium is generally adjusted to 6.8±0.1.

Bacterial strains can be isolated from samples by plating a specificvolume (e.g., about 1 μl) of the fecal material with directly on YCFAGmedium. After a specific incubation time (e.g., 12 h to 16 h) at 37° C.in an anaerobic tent (approximately 80% N₂, 12% CO₂, and 8% H₂), 500translucent colonies per sample are selected and subcultured on freshplates (50 per plate in a grid-like fashion). After continued growth,bacterial colonies can presumptively be identified based on morphology.The majority of colonies (e.g., 95%) are disregarded, with the remainingcolonies being submitted for purification and further analysis includingGram staining. Colonies of the isolated strains (i.e., isolate samples)can be routinely maintained by growing colonies for 16 to 18 h at 37° C.in 7.5-ml aliquots of M2GSC medium and maintained anaerobically usingO₂-free CO₂. Isolate samples obtained and isolated from fecal matter canalso be subjected to metagenomic sequencing in order to profile theentire bacterial community present.

Isolated strains disclosed herein have been deposited in the DSM LeibnizInstitute DSMZ—German Collection of Microorganisms and Cell Cultures,Inhoffenstr. 7B, D-38124 Braunschweig, Germany in accordance with andunder the provisions of the Budapest Treaty for the Deposit ofMicroorganisms, i.e., they will be stored with all the care necessary tokeep them viable and uncontaminated for a period of at least five yearsafter the most recent request for the furnishing of a sample of thedeposits, and in any case, for a period of at least 30 (thirty) yearsafter the date of deposit or for the enforceable life of any patentwhich may issue disclosing the cultures plus five years after the lastrequest for a sample from the deposit. The strains were tested by theDSMZ and determined to be viable. The DSMZ has assigned the followingdeposit accession numbers to the strains: Coprococcus comes ST3 (DSM33176), Bacteroides faecis (DSM 33177), Bacteroides thetaiotaomicron(DSM 33178), Bifidobacterium Longum ST27 (DSM 33179), Blautia productaST4 (DSM 33180), Faecalibacterium prausnitzii ST38 (DSM 33185),Faecalibacterium prausnitzii ST23 (DSM 33186), Lactobacillus crispatusST100 (DSM 33187), Dorea longicatena (DSM 33188), Faecalibacteriumprausnitzii ST27(DSM 33190), Faecalibacterium prausnitzii ST58 (DSM33191), Akkermansia muciniphila ST7 (DSM 33213), all of which weredeposited on Jun. 27, 2019. The depositor acknowledges the duty toreplace the deposits should the depository be unable to furnish a samplewhen requested, due to the condition of the deposits. All restrictionson the availability to the public of the subject culture deposits willbe irrevocably removed upon the granting of a patent disclosing them.The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject matter disclosed herein in derogation of patent rights grantedby governmental action.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1 Preparation of the Anaerobic Chamber for Isolation ofAnaerobic Bacterial Colonies

This example describes illustrative steps conducted in preparation tohandling and isolating anaerobic bacterial strains within an anaerobicchamber.

All sterile and pre-reduced consumable plastics and culture media wereplaced into the anaerobic chamber at least one day prior to utilizationto allow for full oxygen reduction. Further, all tubes, vials, andflasks were left in the anaerobic chamber with open lids in order toallow for gas exchange and reduction over night.

The following consumables were placed into the anaerobic chamber atleast one day prior to use: >100 mL PBS +0.5 mg/mL cysteine (PBS+Cys); 1mL filter tips, 1.5 mL tubes, 2 mL screw cap cryovials containing 0.75mL of 50% glycerol in PBS.

Agar and broth culture media are prepared and transferred to theanaerobic chamber at least one day prior to use. The anaerobic mediacontained either 1 mM Na₂S.9H₂O or 0.5 mg/mL cysteine as reducing agentsto promote growth of strict anaerobes. Agar plates were poured insidethe anaerobic chamber to prevent the oxidation of media components afterautoclaving.

Example 2 Growth, Isolation, and Selection of Liquid Culture Colonies

This example describes the culture of obligatory and facultativeanaerobic bacterial strains that were derived from human sample onselective media. Upon subculturing, the colonies were transferred to aliquid medium and subsequently prepared for PCR and sequencing. Colonieswere also preserved as glycerol stocks.

Human samples were collected in anaerobic transport media (AnaerobicSystems As-915) or in fecal collection vials sealed within a plastic bagcontaining an anaerobic atmosphere generating system (e.g. AnaeroPouchThermo Fisher R686001). All samples were immediately transferred into ananaerobic chamber to minimize transit time and potential oxygen exposureto ensure viability of the anaerobic strains.

Serial dilution tubes were prepared by aliquoting 0.9 mL of PBS+Cys into13 tubes (with a volume of 1.5 mL). Using a disposable spatula or loop,20-30 mg of sample is transferred into a first 1.5 mL tube containing0.9 mL of PBS+Cys. The resulting mixture was vortexed for approximately30 seconds and 0.1 mL of the resulting, homogenous solution istransferred into the second 1.5 mL tube containing 0.9 mL of PBS+Cys.This step was then repeated until all 13 tubes contain serial dilutions(10⁰-10⁻¹² dilutions in vials 1-13) of the sample.

Using a disposable hockey stick spreader, ˜0.1 mL from the sample tubescontaining the dilutions 10⁻⁵ to 10⁻¹² (vials 6-13) were added toseparate agar plates containing the selective agar growth media. Theagar plates were then sealed with parafilm to prevent evaporation andplaced into an incubator for 72 hours at 37° C. Colonies that fit atarget's colony morphology were identified, and placed on a new,pre-reduced agar plate for isolation. The agar plates were sealed withparafilm and placed into an incubator for another 72 hours at 37° C.

For instance, the target morphology of Lactobacillus johnsonii colonieswas circular white colonies with an entire margin and convex elevation,and with a diameter of 2-3 mm. The target morphology of Faecalibacteriumprausnitzii colonies appeared as circular shaped and with a tan color,with an entire margin and flat to umbonate elevation, and with adiameter of 2-3 mm. The target morphology of Akkermansia muciniphilacolonies appeared as circular shaped and opague, with an entire marginand convex elevation, and with a diameter of 1-2 mm.

The isolated colonies were transferred into liquid media by pickingtarget isolated colonies from culture plates and resuspending thecolonies in 1 mL of pre-reduced liquid broth. Positive and negativecontrols of target organisms are inoculated in parallel to compare forgrowth and monitor for contamination, respectively. All liquid colonysamples are then incubated for 72 hours at 37° C.

Using the positive and negative controls, positive match broth cultureswere identified. Glycerol stocks of positive match broth cultures wereprepared by transferring 0.75 mL of the broth culture solution into a 2mL cryotube containing 0.75 mL of 50% glycerol in PBS. Sealed cryotubesamples were then removed from the anaerobic chamber and stored at −80°C. The remaining broth culture samples were used for isolateidentification using 16S-based PCR as described in the example below(EXAMPLE 3).

Example 3 16S-Based PCR for Isolate Identification

This example describes a method for conducting 16S-based PCR for isolateidentification.

Broth culture samples were centrifuged to form cell pellets and theresulting supernatant is carefully removed to leave the formed cellpellet intact. The cell pellet is then resuspended in 0.5-1 mL ultrapurewater.

The PCR Mastermix for a final reaction volume of 50 μL was preparedusing the following PCR components (NEB E5000S) and volumes: 10×Buffer(5 μL), 10 mM dNTPs (1 μL), 10 μM 27F Forward Primer (1 μL), 10 μM 1492RReverse Primer (1 μL), Tag Polymerase (0.25 μL), and sterile water(40.25 μL). The PCR Mastermix (48.5 μL) and 1.5 μL of resuspendedbacterial cells are placed into a 0.2 mL PCR strip tube and vortexedbefore the PCR reaction samples are exposed to the followingthermocycler protocol:

Step Temp Time 1 95° C.  2 min 2 95° C. 30 sec 3 50° C. 30 sec 4 68° C.1 min 30 sec (30 repeats of steps 2-4) 5 72° C.  5 min 6  4° C. HOLD

Upon completion of the PCR reactions, samples can be submitted forSanger sequencing using GENEWIZ or a comparable vendor.

Example 4 Listing and Identification of Refined and Isolated BacterialStrains

This example shows all isolates (isolate numbers 1-251) that wereproduced using the isolation protocol as described above (EXAMPLE 2).All isolates were further identified in terms of their taxonomy usingthe above described protocol for 16S-based PCR (EXAMPLE 3).

Overall, 251 isolated bacterial strains (e.g., isolates) were identifiedand characterized. Fecal samples were obtained from healthy femalechildren (human), healthy male and female adults (human), and fromfemale C57/blk6 mice (mus musculus).

Table 3 below shows the isolate numbers, species, sample sources, andisolation media for all isolated strains 1-251.

Table 4 below shows the 16S-based PCR results for all isolates,including quality score and identity.

Table 5 below shows the taxonomic identification of all bacterialisolates 1-251, including phylum, class, family, genus, and species.

TABLE 3 Species, Source, and Isolation Media Information for IsolateSamples (Strains) 1-251 Isolate Number Species Source Source IDIsolation Media 1 Akkermansia muciniphila Human - fecal healthy child ♀Mucin Minimal Media 2 Akkermansia muciniphila Human - fecal healthychild ♀ Mucin Minimal Media 3 Akkermansia muciniphila Human - fecalhealthy child ♀ Mucin Minimal Media 4 Akkermansia muciniphila Human -fecal healthy adult ♀ Mucin Minimal Media 5 Akkermansia muciniphila Musmusculus - fecal C57/blk6 ♀ Mucin Minimal Media 6 Akkermansiamuciniphila Mus musculus - fecal C57/blk6 ♀ Mucin Minimal Media 7Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀ Mucin MinimalMedia 8 Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀ MucinMinimal Media 9 Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀Mucin Minimal Media 10 Akkermansia muciniphila Mus musculus - fecalC57/blk6 ♀ Mucin Minimal Media 11 Akkermansia muciniphila Mus musculus -fecal C57/blk6 ♀ Mucin Minimal Media 12 Akkermansia muciniphila Musmusculus - fecal C57/blk6 ♀ Mucin Minimal Media 13 Akkermansiamuciniphila Mus musculus - fecal C57/blk6 ♀ Mucin Minimal Media 14Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀ Mucin MinimalMedia 15 Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀ MucinMinimal Media 16 Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀Mucin Minimal Media 17 Akkermansia muciniphila Mus musculus - fecalC57/blk6 ♀ Mucin Minimal Media 18 Akkermansia muciniphila Mus musculus -fecal C57/blk6 ♀ Mucin Minimal Media 19 Akkermansia muciniphila Musmusculus - fecal C57/blk6 ♀ Mucin Minimal Media 20 Akkermansiamuciniphila Mus musculus - fecal C57/blk6 ♀ Mucin Minimal Media 21Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀ Mucin MinimalMedia 22 Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀ MucinMinimal Media 23 Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀Mucin Minimal Media 24 Akkermansia muciniphila Mus musculus - fecalC57/blk6 ♀ Mucin Minimal Media 25 Akkermansia muciniphila Mus musculus -fecal C57/blk6 ♀ Mucin Minimal Media 26 Akkermansia muciniphila Musmusculus - fecal C57/blk6 ♀ Mucin Minimal Media 27 Akkermansiamuciniphila Mus musculus - fecal C57/blk6 ♀ Mucin Minimal Media 28Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀ Mucin MinimalMedia 29 Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀ MucinMinimal Media 30 Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀Mucin Minimal Media 31 Akkermansia muciniphila Mus musculus - fecalC57/blk6 ♀ Mucin Minimal Media 32 Akkermansia muciniphila Mus musculus -fecal C57/blk6 ♀ Mucin Minimal Media 33 Akkermansia muciniphila Musmusculus - fecal C57/blk6 ♀ Mucin Minimal Media 34 Akkermansiamuciniphila Mus musculus - fecal C57/blk6 ♀ Mucin Minimal Media 35Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀ Mucin MinimalMedia 36 Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀ MucinMinimal Media 37 Akkermansia muciniphila Mus musculus - fecal C57/blk6 ♀Mucin Minimal Media 38 Akkermansia muciniphila Mus musculus - fecalC57/blk6 ♀ Mucin Minimal Media 39 Akkermansia muciniphila Mus musculus -fecal C57/blk6 ♀ Mucin Minimal Media 40 Akkermansia muciniphila Musmusculus - fecal C57/blk6 ♀ Mucin Minimal Media 41 Anaerostipes hadrusHuman - fecal healthy adult ♀ Yeast Fatty Acid Casitone Media 42Anaerotignum lactatifermentans Human - fecal healthy adult ♂ Yeast FattyAcid Casitone Media 43 Bacteroides caccae Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 44 Bacteroides caccae Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 45 Bacteroides doreiHuman - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 46Bacteroides dorei Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 47 Bacteroides dorei Human - fecal healthy child ♀ MucinMinimal Media 48 Bacteroides faecis Human - fecal healthy adult ♀ MucinMinimal Media 49 Bacteroides faecis Human - fecal healthy adult ♀ MucinMinimal Media 50 Bacteroides faecis Human - fecal healthy adult ♀ YeastFatty Acid Casitone Media 51 Bacteroides faecis Human - fecal healthyadult ♀ Yeast Fatty Acid Casitone Media 52 Bacteroides faecis Human -fecal healthy adult ♀ Yeast Fatty Acid Casitone Media 53 Bacteroidesfaecis Human - fecal healthy adult ♀ Yeast Fatty Acid Casitone Media 54Bacteroides faecis Human - fecal healthy adult ♀ Yeast Fatty AcidCasitone Media 55 Bacteroides faecis Human - fecal healthy child ♀ YeastFatty Acid Casitone Media 56 Bacteroides faecis Human - fecal healthychild ♀ Yeast Fatty Acid Casitone Media 57 Bacteroides faecis Human -fecal healthy child ♀ Yeast Fatty Acid Casitone Media 58 Bacteroidesfinegoldii Human - fecal healthy adult ♀ Yeast Fatty Acid Casitone Media59 Bacteroides fragilis Human - fecal healthy adult ♂ Yeast Fatty AcidCasitone Media 60 Bacteroides fragilis Human - fecal healthy adult ♂Yeast Fatty Acid Casitone Media 61 Bacteroides fragilis Human - fecalhealthy adult ♂ Yeast Fatty Acid Casitone Media 62 Bacteroides fragilisHuman - fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 63Bacteroides fragilis Human - fecal healthy adult ♂ Yeast Fatty AcidCasitone Media 64 Bacteroides fragilis Human - fecal healthy adult ♂Yeast Fatty Acid Casitone Media 65 Bacteroides fragilis Human - fecalhealthy adult ♂ Yeast Fatty Acid Casitone Media 66 Bacteroides fragilisHuman - fecal healthy adult ♂ Mucin Minimal Media 67 Bacteroidesfragilis Human - fecal healthy adult ♂ Mucin Minimal Media 68Bacteroides fragilis Human - fecal healthy child ♀ Mucin Minimal Media69 Bacteroides fragilis Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 70 Bacteroides fragilis Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 71 Bacteroides fragilis Human - fecalhealthy adult ♀ Yeast Fatty Acid Casitone Media 72 Bacteroides fragilisHuman - fecal healthy adult ♀ Yeast Fatty Acid Casitone Media 73Bacteroides intestinalis Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 74 Bacteroides stercoris Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 75 Bacteroides stercoris Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 76 Bacteroides stercorisHuman - fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 77Bacteroides stercoris Human - fecal healthy adult ♂ Yeast Fatty AcidCasitone Media 78 Bacteroides stercoris Human - fecal healthy adult ♂Yeast Fatty Acid Casitone Media 79 Bacteroides stercoris Human - fecalhealthy adult ♂ Yeast Fatty Acid Casitone Media 80 Bacteroides stercorisHuman - fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 81Bacteroides stercoris Human - fecal healthy adult ♀ Yeast Fatty AcidCasitone Media 82 Bacteroides thetaiotaomicron Human - fecal healthyadult ♂ Yeast Fatty Acid Casitone Media 83 Bacteroides thetaiotaomicronHuman - fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 84Bacteroides thetaiotaomicron Human - fecal healthy child ♀ Yeast FattyAcid Casitone Media 85 Bacteroides thetaiotaomicron Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 86 Bacteroidesthetaiotaomicron Human - fecal healthy child ♀ Yeast Fatty Acid CasitoneMedia 87 Bacteroides thetaiotaomicron Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 88 Bacteroides thetaiotaomicron Musmusculus - fecal C57/blk6 ♀ Mucin Minimal Media 89 Bacteroidesthetaiotaomicron Human - fecal healthy adult ♀ Yeast Fatty Acid CasitoneMedia 90 Bacteroides thetaiotaomicron Human - fecal healthy adult ♀Mucin Minimal Media 91 Bacteroides thetaiotaomicron Human - fecalhealthy adult ♀ Yeast Fatty Acid Casitone Media 92 Bacteroides uniformisHuman - fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 93Bacteroides uniformis Human - fecal healthy adult ♂ Yeast Fatty AcidCasitone Media 94 Bacteroides uniformis Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 95 Bacteroides vulgatus Human - fecalhealthy adult ♀ Yeast Fatty Acid Casitone Media 96 Bacteroides vulgatusHuman - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 97Bacteroides vulgatus Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 98 Bacteroides vulgatus Human - fecal healthy adult ♂Yeast Fatty Acid Casitone Media 99 Bacteroides vulgatus Human - fecalhealthy adult ♂ Yeast Fatty Acid Casitone Media 100 Bacteroides vulgatusHuman - fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 101Bacteroides vulgatus Human - fecal healthy adult ♂ Yeast Fatty AcidCasitone Media 102 Bacteroides vulgatus Human - fecal healthy adult ♂Yeast Fatty Acid Casitone Media 103 Bacteroides vulgatus Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 104 Bacteroides vulgatusHuman - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 105Bacteroides vulgatus Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 106 Bacteroides vulgatus Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 107 Bacteroides vulgatus Human - fecalhealthy adult ♀ Yeast Fatty Acid Casitone Media 108 Bacteroides vulgatusHuman - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 109Bacteroides xylanisolvens Human - fecal healthy adult ♂ Mucin MinimalMedia 110 Bacteroides xylanisolvens Human - fecal healthy child ♀ YeastFatty Acid Casitone Media 111 Bifidobacterium faecale Human - fecalhealthy child ♀ Bifidobacterium Selective 112 Bifidobacterium faecaleHuman - fecal healthy child ♀ Bifidobacterium Selective 113Bifidobacterium longum Human - fecal healthy adult ♀ BifidobacteriumSelective 114 Bifidobacterium stercoris Human - fecal healthy adult ♂Bifidobacterium Selective 115 Bifidobacterium stercoris Human - fecalhealthy adult ♂ Bifidobacterium Selective 116 Blautia faecis Human -fecal healthy adult ♀ Yeast Fatty Acid Casitone Media 117 Blautia faecisHuman - fecal healthy adult ♀ Yeast Fatty Acid Casitone Media 118Blautia gnavus Human - fecal healthy adult ♂ Yeast Fatty Acid CasitoneMedia 119 Blautia luti Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 120 Blautia obeum Human - fecal healthy adult ♀ YeastFatty Acid Casitone Media 121 Blautia obeum Human - fecal healthy child♀ Yeast Fatty Acid Casitone Media 122 Blautia obeum Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 123 Blautia obeumHuman - fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 124Blautia obeum Human - fecal healthy child ♀ Yeast Fatty Acid CasitoneMedia 125 Blautia obeum Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 126 Blautia product Human - fecal healthy adult ♀ YeastFatty Acid Casitone Media 127 Blautia product Human - fecal healthyadult ♀ Yeast Fatty Acid Casitone Media 128 Blautia product Human -fecal healthy adult ♀ Yeast Fatty Acid Casitone Media 129 Blautiaproduct Human - fecal healthy adult ♀ Yeast Fatty Acid Casitone Media130 Blautia product Human - fecal healthy adult ♀ Yeast Fatty AcidCasitone Media 131 Blautia product Human - fecal healthy adult ♀ YeastFatty Acid Casitone Media 132 Blautia product Human - fecal healthyadult ♀ Yeast Fatty Acid Casitone Media 133 Blautia product Human -fecal healthy child ♀ Mucin Minimal Media 134 Blautia product Human -fecal healthy adult ♀ Mucin Minimal Media 135 Blautia product Human -fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 136 Blautiastercoris Human - fecal healthy adult ♂ Yeast Fatty Acid Casitone Media137 Blautia stercoris Human - fecal healthy adult ♂ Yeast Fatty AcidCasitone Media 138 Blautia torque Human - fecal healthy adult ♀ YeastFatty Acid Casitone Media 139 Blautia wexlerae Human - fecal healthychild ♀ Yeast Fatty Acid Casitone Media 140 Blautia wexlerae Human -fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 141 Blautiawexlerae Human - fecal healthy adult ♂ Mucin Minimal Media 142 Blautiawexlerae Human - fecal healthy adult ♂ Yeast Fatty Acid Casitone Media143 Blautia wexlerae Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 144 Blautia wexlerae Human - fecal healthy child ♀ MucinMinimal Media 145 Blautia wexlerae Human - fecal healthy child ♀ YeastFatty Acid Casitone Media 146 Collinsella aerofaciens Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 147 Coprococcus comesHuman - fecal healthy adult ♀ Yeast Fatty Acid Casitone Media 148Coprococcus comes Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 149 Coprococcus comes Human - fecal healthy child ♀ YeastFatty Acid Casitone Media 150 Coprococcus comes Human - fecal healthychild ♀ Yeast Fatty Acid Casitone Media 151 Coprococcus comes Human -fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 152 Coprococcuscomes Human - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 153Coprococcus comes Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 154 Coprococcus eutactus Human - fecal healthy adult ♂Yeast Fatty Acid Casitone Media 155 Dorea formicigenerans Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 156 Doreaformicigenerans Human - fecal healthy child ♀ Yeast Fatty Acid CasitoneMedia 157 Dorea formicigenerans Human - fecal healthy child ♀ YeastFatty Acid Casitone Media 158 Dorea longicatena Human - fecal healthyadult ♂ Yeast Fatty Acid Casitone Media 159 Dorea longicatena Human -fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 160 Dorealongicatena Human - fecal healthy adult ♂ Yeast Fatty Acid CasitoneMedia 161 Eisenbergiella massiliensis Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 162 Erysipelatoclostridium ramosumHuman - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 163Erysipelatoclostridium ramosum Human - fecal healthy child ♀ Yeast FattyAcid Casitone Media 164 Erysipelatoclostridium ramosum Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 165Erysipelatoclostridium ramosum Human - fecal healthy adult ♀ Yeast FattyAcid Casitone Media 166 Erysipelatoclostridium ramosum Human - fecalhealthy adult ♀ Yeast Fatty Acid Casitone Media 167Erysipelatoclostridium ramosum Human - fecal healthy adult ♀ Yeast FattyAcid Casitone Media 168 Erysipelatoclostridium ramosum Human - fecalhealthy adult ♀ Yeast Fatty Acid Casitone Media 169 Escherichiafergusonii Human - fecal healthy adult ♀ Yeast Fatty Acid Casitone Media170 Escherichia fergusonii Human - fecal healthy adult ♀ Yeast FattyAcid Casitone Media 171 Escherichia fergusonii Human - fecal healthyadult ♀ Yeast Fatty Acid Casitone Media 172 Escherichia fergusoniiHuman - fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 173Escherichia fergusonii Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 174 Escherichia fergusonii Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 175 Escherichia fergusonii Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 176 Escherichiafergusonii Human - fecal healthy child ♀ Yeast Fatty Acid Casitone Media177 Escherichia fergusonii Human - fecal healthy child ♀ Yeast FattyAcid Casitone Media 178 Escherichia fergusonii Human - fecal healthychild ♀ Yeast Fatty Acid Casitone Media 179 Escherichia fergusoniiHuman - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 180Escherichia fergusonii Human - fecal healthy adult ♂ Yeast Fatty AcidCasitone Media 181 Escherichia fergusonii Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 182 Eubacterium hallii Human - fecalhealthy adult ♀ Yeast Fatty Acid Casitone Media 183 Eubacteriumventriosum Human - fecal healthy child ♀ Yeast Fatty Acid Casitone Media184 Faecalibacterium prausnitzii Human - fecal healthy child ♀ YeastFatty Acid Casitone Media 185 Faecalibacterium prausnitzii Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 186 Faecalibacteriumprausnitzii Human - fecal healthy adult ♂ Yeast Fatty Acid CasitoneMedia 187 Faecalibacterium prausnitzii Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 188 Faecalibacterium prausnitzii Human -fecal healthy adult ♂ Yeast Fatty Acid Casitone Media 189Faecalibacterium prausnitzii Human - fecal healthy child ♀ Yeast FattyAcid Casitone Media 190 Faecalibacterium prausnitzii Human - fecalhealthy adult ♂ Yeast Fatty Acid Casitone Media 191 Faecalibacteriumprausnitzii Human - fecal healthy adult ♂ Yeast Fatty Acid CasitoneMedia 192 Faecalibacterium prausnitzii Human - fecal healthy adult ♂Yeast Fatty Acid Casitone Media 193 Faecalibacterium prausnitzii Human -fecal healthy child ♀ Yeast Fatty Acid Casitone Media 194Faecalibacterium prausnitzii Human - fecal healthy child ♀ Yeast FattyAcid Casitone Media 195 Faecalibacterium prausnitzii Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 196 Faecalibacteriumprausnitzii Human - fecal healthy child ♀ Yeast Fatty Acid CasitoneMedia 197 Faecalibacterium prausnitzii Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 198 Faecalibacterium prausnitzii Human -fecal healthy child ♀ Yeast Fatty Acid Casitone Media 199Faecalibacterium prausnitzii Human - fecal healthy child ♀ Yeast FattyAcid Casitone Media 200 Holdemanella biforme Human - fecal healthy child♀ Yeast Fatty Acid Casitone Media 201 Holdemanella biforme Human - fecalhealthy adult ♂ Yeast Fatty Acid Casitone Media 202 Holdemanella biformeHuman - fecal healthy adult ♀ Yeast Fatty Acid Casitone Media 203Hungatella effluvia Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 204 Hungatella hathewayi Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 205 Lachnoclostridium aerotoleransHuman - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 206Lachnoclostridium aerotolerans Human - fecal healthy child ♀ Yeast FattyAcid Casitone Media 207 Lachnoclostridium aldenense Human - fecalhealthy adult ♂ Yeast Fatty Acid Casitone Media 208 Lachnoclostridiumasparagiforme Human - fecal healthy child ♀ Yeast Fatty Acid CasitoneMedia 209 Lachnoclostridium bolteae Human - fecal healthy child ♀ YeastFatty Acid Casitone Media 210 Lachnoclostridium lavalense Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 211 Lachnoclostridiumsymbiosum Human - fecal healthy adult ♂ Yeast Fatty Acid Casitone Media212 Lachnospira pectinoschiza Human - fecal healthy adult ♀ Yeast FattyAcid Casitone Media 213 Lactobacillus coleohominis Human - vaginalhealthy adult ♀ de Man Rogosa Sharpe Media 214 Lactobacillus crispatusHuman - vaginal healthy adult ♀ de Man Rogosa Sharpe Media 215Lactobacillus crispatus Human - vaginal healthy adult ♀ de Man RogosaSharpe Media 216 Lactobacillus crispatus Human - vaginal healthy adult ♀de Man Rogosa Sharpe Media 217 Lactobacillus gasseri Mus musculus -fecal C57/blk6 ♀ de Man Rogosa Sharpe Media 218 Lactobacillus jenseniiHuman - vaginal healthy adult ♀ de Man Rogosa Sharpe Media 219Lactobacillus jensenii Human - vaginal healthy adult ♀ de Man RogosaSharpe Media 220 Lactobacillus jensenii Human - vaginal healthy adult ♀de Man Rogosa Sharpe Media 221 Lactobacillus jensenii Human - vaginalhealthy adult ♀ de Man Rogosa Sharpe Media 222 Lactobacillus jenseniiHuman - vaginal healthy adult ♀ de Man Rogosa Sharpe Media 223Lactobacillus johnsonii Mus musculus - fecal C57/blk6 ♀ de Man RogosaSharpe Media 224 Lactobacillus johnsonii Mus musculus - fecal C57/blk6 ♀de Man Rogosa Sharpe Media 225 Lactobacillus johnsonii Mus musculus -fecal C57/blk6 ♀ de Man Rogosa Sharpe Media 226 Lactobacillus johnsoniiMus musculus - fecal C57/blk6 ♀ de Man Rogosa Sharpe Media 227Lactonifactor longoviformis Human - fecal healthy child ♀ Yeast FattyAcid Casitone Media 228 Longibaculum muris Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 229 Longibaculum muris Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 230 Longibaculum murisHuman - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 231Longibaculum muris Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 232 Muribaculum intestinale Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 233 Oscillibacter ruminantium Human -fecal healthy child ♀ Yeast Fatty Acid Casitone Media 234 Oscillibacterruminantium Human - fecal healthy child ♀ Yeast Fatty Acid CasitoneMedia 235 Oscillibacter ruminantium Human - fecal healthy child ♀ YeastFatty Acid Casitone Media 236 Oscillibacter ruminantium Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 237 Parabacteroidesdistasonis Human - fecal healthy child ♀ Yeast Fatty Acid Casitone Media238 Parabacteroides merdae Human - fecal healthy child ♀ Yeast FattyAcid Casitone Media 239 Parabacteroides merdae Human - fecal healthychild ♀ Yeast Fatty Acid Casitone Media 240 Parabacteroides merdaeHuman - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 241Parabacteroides merdae Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 242 Parabacteroides merdae Human - fecal healthy child ♀Yeast Fatty Acid Casitone Media 243 Propionibacterium acnes Musmusculus - fecal C57/blk6 ♀ Mucin Minimal Media 244 Roseburiainulinivorans Human - fecal healthy child ♀ Yeast Fatty Acid CasitoneMedia 245 Roseburia inulinivorans Human - fecal healthy child ♀ YeastFatty Acid Casitone Media 246 Roseburia inulinivorans Human - fecalhealthy child ♀ Yeast Fatty Acid Casitone Media 247 Ruminococcus bromiiHuman - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 248Shigella flexneri Human - fecal healthy child ♀ Yeast Fatty AcidCasitone Media 249 Staphylococcus epidermidis Human - fecal healthyadult ♀ Yeast Fatty Acid Casitone Media 250 Staphylococcus warneriHuman - fecal healthy child ♀ Yeast Fatty Acid Casitone Media 251Sutterella wadsworthensis Mus musculus - fecal C57/blk6 ♀ Mucin MinimalMedia

TABLE 4 16S-Based PCR Results Obtained for Isolate Samples 1-251Continuous Quality Reading Isolate Score Length Query Number (QS) (CRL)cover Identification 1 45 993 0.9 0.99 2 48 1034 0.97 0.98 3 46 10180.92 0.98 4 47 1001 0.95 0.98 5 47 946 0.96 0.98 6 48 931 0.95 0.99 7 48919 0.95 0.99 8 46 966 0.95 0.98 9 45 921 0.95 0.97 10 45 982 0.96 0.9811 49 919 0.96 0.99 12 50 913 0.95 0.99 13 46 942 0.96 0.98 14 49 9440.97 0.99 15 49 903 0.95 0.99 16 48 936 0.96 0.99 17 44 932 0.95 0.98 1845 923 0.95 0.98 19 48 898 0.96 0.98 20 47 937 0.94 0.99 21 45 949 0.950.98 22 47 936 0.95 0.98 23 45 931 0.95 0.98 24 48 952 0.95 0.97 25 47921 0.96 0.99 26 48 954 0.96 0.99 27 48 952 0.96 0.99 28 47 937 0.960.98 29 43 895 0.95 0.97 30 46 968 0.96 0.98 31 47 950 0.95 0.99 32 46942 0.96 0.98 33 42 918 0.96 0.96 34 45 920 0.96 0.98 35 48 914 0.950.99 36 47 934 0.95 0.98 37 47 937 0.95 0.99 38 49 907 0.95 0.98 39 45918 0.95 0.98 40 45 907 0.94 0.98 41 39 933 0.89 0.96 42 47 995 0.960.94 43 50 1018 0.97 0.99 44 50 1016 0.98 0.98 45 48 999 0.96 0.99 46 511003 0.97 0.99 47 41 993 0.97 0.99 48 47 996 0.94 0.98 49 48 992 0.960.98 50 45 988 0.93 0.97 51 47 993 0.94 0.98 52 47 999 0.94 0.98 53 48960 0.96 0.98 54 46 992 0.89 0.98 55 43 966 0.96 0.85 56 37 269 0.990.88 57 49 1014 0.97 0.99 58 43 958 0.87 0.97 59 49 1002 0.96 0.98 60 49991 0.97 0.98 61 48 994 0.97 0.98 62 50 991 0.97 0.98 63 46 992 0.970.98 64 50 994 0.97 0.98 65 49 979 0.96 0.98 66 50 996 0.96 0.99 67 50987 0.97 0.98 68 48 1006 0.96 0.98 69 48 1016 0.95 0.97 70 40 799 0.780.98 71 49 998 0.97 0.98 72 49 1028 0.98 0.98 73 50 991 0.97 0.99 74 471033 0.97 0.99 75 49 999 0.97 0.99 76 47 1048 0.97 0.99 77 50 986 0.980.99 78 49 976 0.97 0.99 79 47 1007 0.97 0.97 80 48 1044 0.97 0.97 81 501014 0.97 0.99 82 47 991 0.97 0.98 83 47 977 0.97 0.98 84 47 988 0.970.98 85 48 1000 0.96 0.99 86 50 976 0.97 0.98 87 48 1013 0.96 0.99 88 49918 0.96 0.99 89 49 1016 0.95 0.98 90 46 1009 0.97 0.96 91 46 1002 0.940.97 92 49 1070 0.97 0.99 93 49 989 0.96 0.98 94 46 1015 0.95 0.98 95 42982 0.86 0.98 96 50 987 0.96 0.99 97 49 1009 0.97 0.99 98 49 1010 0.940.98 99 49 1002 0.94 0.98 100 48 1000 0.97 0.97 101 48 996 0.92 0.99 10248 1007 0.96 0.99 103 49 1016 0.94 0.99 104 49 1011 0.96 0.99 105 481016 0.96 0.99 106 49 1011 0.97 0.99 107 49 1031 0.97 0.99 108 46 10390.9 0.99 109 48 1014 0.97 0.99 110 48 1003 0.97 0.99 111 43 975 0.940.96 112 50 997 0.94 0.99 113 46 1010 0.88 0.98 114 49 1042 0.97 0.98115 50 1021 0.97 0.98 116 45 990 0.83 0.98 117 39 1004 0.79 0.98 118 471064 0.93 0.99 119 47 1019 0.97 0.96 120 43 934 0.91 0.94 121 45 9480.95 0.89 122 38 939 0.96 0.8 123 49 990 0.96 0.97 124 47 1043 0.91 0.98125 47 982 0.93 0.98 126 46 975 0.93 0.96 127 46 976 0.95 0.95 128 47972 0.96 0.97 129 46 988 0.91 0.97 130 47 1018 0.91 0.98 131 48 993 0.940.97 132 46 993 0.89 0.97 133 25 144 0.91 0.83 134 48 1034 0.98 0.98 13545 1054 0.94 0.98 136 48 1006 0.97 0.94 137 50 975 0.95 0.95 138 44 9760.88 0.98 139 50 961 0.96 0.98 140 47 978 0.97 0.97 141 48 1007 0.970.97 142 47 970 0.97 0.97 143 47 948 0.97 0.97 144 47 988 0.97 0.97 14548 974 0.96 0.98 146 48 1019 0.95 0.98 147 43 932 0.91 0.96 148 46 9820.97 0.89 149 25 535 0.93 0.9 150 32 373 0.95 0.93 151 48 1030 0.97 0.98152 43 975 0.94 0.98 153 49 1040 0.96 0.98 154 49 1013 0.97 0.98 155 48997 0.97 0.97 156 49 1018 0.96 0.97 157 48 1026 0.97 0.97 158 48 9950.89 0.98 159 49 1011 0.9 0.99 160 48 1043 0.89 0.99 161 36 345 0.97 0.9162 49 991 0.98 0.99 163 49 1015 0.96 0.99 164 48 991 0.96 0.99 165 491028 0.97 0.98 166 48 1024 0.97 0.98 167 49 1025 0.98 0.98 168 48 9910.97 0.98 169 46 946 0.95 0.97 170 44 924 0.92 0.97 171 47 936 0.96 0.97172 48 1038 0.97 0.98 173 48 963 0.97 0.98 174 48 1041 0.97 0.99 175 481003 0.97 0.99 176 49 975 0.96 0.99 177 50 976 0.98 0.99 178 49 999 0.970.98 179 49 998 0.94 0.98 180 48 1046 0.97 0.98 181 44 962 0.94 0.98 18244 912 0.88 0.96 183 45 984 0.91 0.96 184 48 1040 0.97 0.97 185 46 9440.92 0.97 186 46 922 0.96 0.96 187 48 1015 0.97 0.97 188 46 926 0.970.96 189 49 1002 0.97 0.97 190 46 926 0.97 0.97 191 46 922 0.97 0.96 19246 930 0.96 0.96 193 48 948 0.97 0.96 194 46 1033 0.97 0.96 195 49 10230.97 0.96 196 48 1037 0.97 0.97 197 48 1006 0.97 0.97 198 48 1028 0.970.97 199 49 1008 0.97 0.97 200 47 986 0.97 0.97 201 46 983 0.97 0.97 20243 998 0.96 0.95 203 43 984 0.97 0.98 204 45 978 0.97 0.97 205 31 3270.9 0.88 206 30 656 0.9 0.88 207 45 951 0.85 0.97 208 43 648 0.91 0.95209 46 1017 0.93 0.91 210 44 1024 0.94 0.88 211 48 999 0.96 0.96 212 40943 0.93 0.96 213 42 809 0.87 0.97 214 40 710 0.8 0.97 215 43 852 0.890.98 216 41 771 0.84 0.99 217 39 923 0.95 0.98 218 42 881 0.91 0.96 21941 799 0.85 0.97 220 41 774 0.86 0.96 221 40 794 0.83 0.96 222 43 8690.9 0.97 223 49 902 0.96 0.99 224 47 930 0.96 0.98 225 48 908 0.95 0.98226 44 899 0.94 0.97 227 44 983 0.98 0.92 228 51 998 0.97 0.95 229 50987 0.98 0.95 230 51 984 0.98 0.95 231 50 1029 0.98 0.95 232 47 10020.94 0.84 233 43 986 0.93 0.91 234 39 343 0.93 0.91 235 38 813 0.9 0.9236 50 999 0.96 0.95 237 28 867 0.94 0.81 238 48 990 0.97 0.99 239 49978 0.97 0.99 240 48 1019 0.96 0.99 241 49 1007 0.97 0.99 242 49 10250.96 0.99 243 48 868 0.96 0.95 244 45 966 0.96 0.96 245 46 974 0.97 0.97246 48 981 0.97 0.98 247 46 975 0.96 0.96 248 48 954 0.97 0.97 249 461006 0.91 0.98 250 50 962 0.97 0.99 251 43 836 0.93 0.97

TABLE 5 Taxonomy Information for All Isolate Samples 1-251 IsolateNumber Phylum Class Family Genus Species 1 VerrucomicrobiaVerrucomicrobiae Akkermansiaceae Akkermansia muciniphila 2Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansia muciniphila3 Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansiamuciniphila 4 Verrucomicrobia Verrucomicrobiae AkkermansiaceaeAkkermansia muciniphila 5 Verrucomicrobia VerrucomicrobiaeAkkermansiaceae Akkermansia muciniphila 6 VerrucomicrobiaVerrucomicrobiae Akkermansiaceae Akkermansia muciniphila 7Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansia muciniphila8 Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansiamuciniphila 9 Verrucomicrobia Verrucomicrobiae AkkermansiaceaeAkkermansia muciniphila 10 Verrucomicrobia VerrucomicrobiaeAkkermansiaceae Akkermansia muciniphila 11 VerrucomicrobiaVerrucomicrobiae Akkermansiaceae Akkermansia muciniphila 12Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansia muciniphila13 Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansiamuciniphila 14 Verrucomicrobia Verrucomicrobiae AkkermansiaceaeAkkermansia muciniphila 15 Verrucomicrobia VerrucomicrobiaeAkkermansiaceae Akkermansia muciniphila 16 VerrucomicrobiaVerrucomicrobiae Akkermansiaceae Akkermansia muciniphila 17Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansia muciniphila18 Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansiamuciniphila 19 Verrucomicrobia Verrucomicrobiae AkkermansiaceaeAkkermansia muciniphila 20 Verrucomicrobia VerrucomicrobiaeAkkermansiaceae Akkermansia muciniphila 21 VerrucomicrobiaVerrucomicrobiae Akkermansiaceae Akkermansia muciniphila 22Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansia muciniphila23 Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansiamuciniphila 24 Verrucomicrobia Verrucomicrobiae AkkermansiaceaeAkkermansia muciniphila 25 Verrucomicrobia VerrucomicrobiaeAkkermansiaceae Akkermansia muciniphila 26 VerrucomicrobiaVerrucomicrobiae Akkermansiaceae Akkermansia muciniphila 27Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansia muciniphila28 Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansiamuciniphila 29 Verrucomicrobia Verrucomicrobiae AkkermansiaceaeAkkermansia muciniphila 30 Verrucomicrobia VerrucomicrobiaeAkkermansiaceae Akkermansia muciniphila 31 VerrucomicrobiaVerrucomicrobiae Akkermansiaceae Akkermansia muciniphila 32Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansia muciniphila33 Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansiamuciniphila 34 Verrucomicrobia Verrucomicrobiae AkkermansiaceaeAkkermansia muciniphila 35 Verrucomicrobia VerrucomicrobiaeAkkermansiaceae Akkermansia muciniphila 36 VerrucomicrobiaVerrucomicrobiae Akkermansiaceae Akkermansia muciniphila 37Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansia muciniphila38 Verrucomicrobia Verrucomicrobiae Akkermansiaceae Akkermansiamuciniphila 39 Verrucomicrobia Verrucomicrobiae AkkermansiaceaeAkkermansia muciniphila 40 Verrucomicrobia VerrucomicrobiaeAkkermansiaceae Akkermansia muciniphila 41 Firmicutes ClostridiaLachnospiracea Anaerostipes hadrus 42 Firmicutes ClostridiaLachnospiraceae Anaerotignum lactatifermentans 43 BacteroidetesBacteroidia Bacteroidaceae Bacteroides caccae 44 BacteroidetesBacteroidia Bacteroidaceae Bacteroides caccae 45 BacteroidetesBacteroidia Bacteroidaceae Bacteroides dorei 46 BacteroidetesBacteroidia Bacteroidaceae Bacteroides dorei 47 BacteroidetesBacteroidia Bacteroidaceae Bacteroides dorei 48 BacteroidetesBacteroidia Bacteroidaceae Bacteroides faecis 49 BacteroidetesBacteroidia Bacteroidaceae Bacteroides faecis 50 BacteroidetesBacteroidia Bacteroidaceae Bacteroides faecis 51 BacteroidetesBacteroidia Bacteroidaceae Bacteroides faecis 52 BacteroidetesBacteroidia Bacteroidaceae Bacteroides faecis 53 BacteroidetesBacteroidia Bacteroidaceae Bacteroides faecis 54 BacteroidetesBacteroidia Bacteroidaceae Bacteroides faecis 55 BacteroidetesBacteroidia Bacteroidaceae Bacteroides faecis 56 BacteroidetesBacteroidia Bacteroidaceae Bacteroides faecis 57 BacteroidetesBacteroidia Bacteroidaceae Bacteroides faecis 58 BacteroidetesBacteroidia Bacteroidaceae Bacteroides finegoldii 59 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 60 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 61 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 62 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 63 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 64 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 65 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 66 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 67 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 68 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 69 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 70 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 71 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 72 BacteroidetesBacteroidia Bacteroidaceae Bacteroides fragilis 73 BacteroidetesBacteroidia Bacteroidaceae Bacteroides intestinalis 74 BacteroidetesBacteroidia Bacteroidaceae Bacteroides stercoris 75 BacteroidetesBacteroidia Bacteroidaceae Bacteroides stercoris 76 BacteroidetesBacteroidia Bacteroidaceae Bacteroides stercoris 77 BacteroidetesBacteroidia Bacteroidaceae Bacteroides stercoris 78 BacteroidetesBacteroidia Bacteroidaceae Bacteroides stercoris 79 BacteroidetesBacteroidia Bacteroidaceae Bacteroides stercoris 80 BacteroidetesBacteroidia Bacteroidaceae Bacteroides stercoris 81 BacteroidetesBacteroidia Bacteroidaceae Bacteroides stercoris 82 BacteroidetesBacteroidia Bacteroidaceae Bacteroides thetaiotaomicron 83 BacteroidetesBacteroidia Bacteroidaceae Bacteroides thetaiotaomicron 84 BacteroidetesBacteroidia Bacteroidaceae Bacteroides thetaiotaomicron 85 BacteroidetesBacteroidia Bacteroidaceae Bacteroides thetaiotaomicron 86 BacteroidetesBacteroidia Bacteroidaceae Bacteroides thetaiotaomicron 87 BacteroidetesBacteroidia Bacteroidaceae Bacteroides thetaiotaomicron 88 BacteroidetesBacteroidia Bacteroidaceae Bacteroides thetaiotaomicron 89 BacteroidetesBacteroidia Bacteroidaceae Bacteroides thetaiotaomicron 90 BacteroidetesBacteroidia Bacteroidaceae Bacteroides thetaiotaomicron 91 BacteroidetesBacteroidia Bacteroidaceae Bacteroides thetaiotaomicron 92 BacteroidetesBacteroidia Bacteroidaceae Bacteroides uniformis 93 BacteroidetesBacteroidia Bacteroidaceae Bacteroides uniformis 94 BacteroidetesBacteroidia Bacteroidaceae Bacteroides uniformis 95 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 96 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 97 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 98 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 99 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 100 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 101 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 102 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 103 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 104 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 105 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 106 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 107 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 108 BacteroidetesBacteroidia Bacteroidaceae Bacteroides vulgatus 109 BacteroidetesBacteroidia Bacteroidaceae Bacteroides xylanisolvens 110 BacteroidetesBacteroidia Bacteroidaceae Bacteroides xylanisolvens 111 ActinobacteriaActinobacteria Bifidobacteriaceae Bifidobacterium faecale 112Actinobacteria Actinobacteria Bifidobacteriaceae Bifidobacterium faecale113 Actinobacteria Actinobacteria Bifidobacteriaceae Bifidobacteriumlongum 114 Actinobacteria Actinobacteria BifidobacteriaceaeBifidobacterium stercoris 115 Actinobacteria ActinobacteriaBifidobacteriaceae Bifidobacterium stercoris 116 Firmicutes ClostridiaLachnospiracea Blautia faecis 117 Firmicutes Clostridia LachnospiraceaBlautia faecis 118 Firmicutes Clostridia Lachnospiracea Blautia gnavus119 Firmicutes Clostridia Lachnospiracea Blautia luti 120 FirmicutesClostridia Lachnospiracea Blautia obeum 121 Firmicutes ClostridiaLachnospiracea Blautia obeum 122 Firmicutes Clostridia LachnospiraceaBlautia obeum 123 Firmicutes Clostridia Lachnospiracea Blautia obeum 124Firmicutes Clostridia Lachnospiracea Blautia obeum 125 FirmicutesClostridia Lachnospiracea Blautia obeum 126 Firmicutes ClostridiaLachnospiracea Blautia producta 127 Firmicutes Clostridia LachnospiraceaBlautia producta 128 Firmicutes Clostridia Lachnospiracea Blautiaproducta 129 Firmicutes Clostridia Lachnospiracea Blautia producta 130Firmicutes Clostridia Lachnospiracea Blautia producta 131 FirmicutesClostridia Lachnospiracea Blautia producta 132 Firmicutes ClostridiaLachnospiracea Blautia producta 133 Firmicutes Clostridia LachnospiraceaBlautia producta 134 Firmicutes Clostridia Lachnospiracea Blautiaproducta 135 Firmicutes Clostridia Lachnospiracea Blautia producta 136Firmicutes Clostridia Lachnospiracea Blautia stercoris 137 FirmicutesClostridia Lachnospiracea Blautia stercoris 138 Firmicutes ClostridiaLachnospiracea Blautia torques 139 Firmicutes Clostridia LachnospiraceaBlautia wexlerae 140 Firmicutes Clostridia Lachnospiracea Blautiawexlerae 141 Firmicutes Clostridia Lachnospiracea Blautia wexlerae 142Firmicutes Clostridia Lachnospiracea Blautia wexlerae 143 FirmicutesClostridia Lachnospiracea Blautia wexlerae 144 Firmicutes ClostridiaLachnospiracea Blautia wexlerae 145 Firmicutes Clostridia LachnospiraceaBlautia wexlerae 146 Actinobacteria Coriobacteriia CoriobacteriaceaeCollinsella aerofaciens 147 Firmicutes Clostridia LachnospiraceaCoprococcus comes 148 Firmicutes Clostridia Lachnospiracea Coprococcuscomes 149 Firmicutes Clostridia Lachnospiracea Coprococcus comes 150Firmicutes Clostridia Lachnospiracea Coprococcus comes 151 FirmicutesClostridia Lachnospiracea Coprococcus comes 152 Firmicutes ClostridiaLachnospiracea Coprococcus comes 153 Firmicutes ClostridiaLachnospiracea Coprococcus comes 154 Firmicutes ClostridiaLachnospiracea Coprococcus eutactus 155 Firmicutes ClostridiaLachnospiracea Dorea formicigenerans 156 Firmicutes ClostridiaLachnospiracea Dorea formicigenerans 157 Firmicutes ClostridiaLachnospiracea Dorea formicigenerans 158 Firmicutes ClostridiaLachnospiracea Dorea longicatena 159 Firmicutes ClostridiaLachnospiracea Dorea longicatena 160 Firmicutes ClostridiaLachnospiracea Dorea longicatena 161 Firmicutes ClostridiaLachnospiraceae Eisenbergiella massiliensis 162 FirmicutesErysipelotrichia Erysipelotrichaceae Erysipelatoclostridium ramosum 163Firmicutes Erysipelotrichia Erysipelotrichaceae Erysipelatoclostridiumramosum 164 Firmicutes Erysipelotrichia ErysipelotrichaceaeErysipelatoclostridium ramosum 165 Firmicutes ErysipelotrichiaErysipelotrichaceae Erysipelatoclostridium ramosum 166 FirmicutesErysipelotrichia Erysipelotrichaceae Erysipelatoclostridium ramosum 167Firmicutes Erysipelotrichia Erysipelotrichaceae Erysipelatoclostridiumramosum 168 Firmicutes Erysipelotrichia ErysipelotrichaceaeErysipelatoclostridium ramosum 169 Proteobacteria GammaproteobacteriaEnterobacteriaceae Escherichia fergusonii 170 ProteobacteriaGammaproteobacteria Enterobacteriaceae Escherichia fergusonii 171Proteobacteria Gammaproteobacteria Enterobacteriaceae Escherichiafergusonii 172 Proteobacteria Gammaproteobacteria EnterobacteriaceaeEscherichia fergusonii 173 Proteobacteria GammaproteobacteriaEnterobacteriaceae Escherichia fergusonii 174 ProteobacteriaGammaproteobacteria Enterobacteriaceae Escherichia fergusonii 175Proteobacteria Gammaproteobacteria Enterobacteriaceae Escherichiafergusonii 176 Proteobacteria Gammaproteobacteria EnterobacteriaceaeEscherichia fergusonii 177 Proteobacteria GammaproteobacteriaEnterobacteriaceae Escherichia fergusonii 178 ProteobacteriaGammaproteobacteria Enterobacteriaceae Escherichia fergusonii 179Proteobacteria Gammaproteobacteria Enterobacteriaceae Escherichiafergusonii 180 Proteobacteria Gammaproteobacteria EnterobacteriaceaeEscherichia fergusonii 181 Proteobacteria GammaproteobacteriaEnterobacteriaceae Escherichia fergusonii 182 Firmicutes ClostridiaEubacteriaceae Eubacterium hallii 183 Firmicutes ClostridiaEubacteriaceae Eubacterium ventriosum 184 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 185 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 186 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 187 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 188 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 189 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 190 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 191 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 192 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 193 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 194 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 195 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 196 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 197 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 198 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 199 Firmicutes ClostridiaRuminococcaceae Faecalibacterium prausnitzii 200 FirmicutesErysipelotrichia Erysipelotrichaceae Holdemanella biforme 201 FirmicutesErysipelotrichia Erysipelotrichaceae Holdemanella biforme 202 FirmicutesErysipelotrichia Erysipelotrichaceae Holdemanella biforme 203 FirmicutesClostridia Clostridiaceae Hungatella effluvii 204 Firmicutes ClostridiaClostridiaceae Hungatella hathewayi 205 Firmicutes ClostridiaLachnospiraceae Lachnoclostridium aerotolerans 206 Firmicutes ClostridiaLachnospiraceae Lachnoclostridium aerotolerans 207 Firmicutes ClostridiaLachnospiraceae Lachnoclostridium aldenense 208 Firmicutes ClostridiaLachnospiraceae Lachnoclostridium asparagiforme 209 FirmicutesClostridia Lachnospiraceae Lachnoclostridium bolteae 210 FirmicutesClostridia Lachnospiraceae Lachnoclostridium lavalense 211 FirmicutesClostridia Lachnospiraceae Lachnoclostridium symbiosum 212 FirmicutesClostridia Lachnospiracea Lachnospira pectinoschiza 213 FirmicutesBacilli Lactobacillaceae Lactobacillus coleohominis 214 FirmicutesBacilli Lactobacillaceae Lactobacillus crispatus 215 Firmicutes BacilliLactobacillaceae Lactobacillus crispatus 216 Firmicutes BacilliLactobacillaceae Lactobacillus crispatus 217 Firmicutes BacilliLactobacillaceae Lactobacillus gasseri 218 Firmicutes BacilliLactobacillaceae Lactobacillus jensenii 219 Firmicutes BacilliLactobacillaceae Lactobacillus jensenii 220 Firmicutes BacilliLactobacillaceae Lactobacillus jensenii 221 Firmicutes BacilliLactobacillaceae Lactobacillus jensenii 222 Firmicutes BacilliLactobacillaceae Lactobacillus jensenii 223 Firmicutes BacilliLactobacillaceae Lactobacillus johnsonii 224 Firmicutes BacilliLactobacillaceae Lactobacillus johnsonii 225 Firmicutes BacilliLactobacillaceae Lactobacillus johnsonii 226 Firmicutes BacilliLactobacillaceae Lactobacillus johnsonii 227 Firmicutes ClostridiaClostridiaceae Lactonifactor longoviformis 228 FirmicutesErysipelotrichia Erysipelotrichaceae Longibaculum muris 229 FirmicutesErysipelotrichia Erysipelotrichaceae Longibaculum muris 230 FirmicutesErysipelotrichia Erysipelotrichaceae Longibaculum muris 231 FirmicutesErysipelotrichia Erysipelotrichaceae Longibaculum muris 232Bacteroidetes Bacteroidia Muribaculaceae Muribaculum intestinale 233Firmicutes Clostridia Oscillospiraceae Oscillibacter ruminantium 234Firmicutes Clostridia Oscillospiraceae Oscillibacter ruminantium 235Firmicutes Clostridia Oscillospiraceae Oscillibacter ruminantium 236Firmicutes Clostridia Oscillospiraceae Oscillibacter ruminantium 237Bacteroidetes Bacteroidia Tannerellaceae Parabacteroides distasonis 238Bacteroidetes Bacteroidia Tannerellaceae Parabacteroides merdae 239Bacteroidetes Bacteroidia Tannerellaceae Parabacteroides merdae 240Bacteroidetes Bacteroidia Tannerellaceae Parabacteroides merdae 241Bacteroidetes Bacteroidia Tannerellaceae Parabacteroides merdae 242Bacteroidetes Bacteroidia Tannerellaceae Parabacteroides merdae 243Actinobacteria Actinobacteria Propionibacteriaceae Cutibacterium acnes244 Firmicutes Clostridia Lachnospiracea Roseburia inulinivorans 245Firmicutes Clostridia Lachnospiracea Roseburia inulinivorans 246Firmicutes Clostridia Lachnospiracea Roseburia inulinivorans 247Firmicutes Clostridia Ruminococcaceae Ruminococcus bromii 248Proteobacteria Gammaproteobacteria Enterobacteriaceae Shigella flexneri249 Firmicutes Bacilli Staphylococcaceae Staphylococcus epidermidis 250Firmicutes Bacilli Staphylococcaceae Staphylococcus warneri 251Proteobacteria Betaproteobacteria Sutterellaceae Sutterellawadsworthensis

Example 5 Characterization of 25 Isolated Bacterial Strains

This example lists examples of isolated strains from Table 3 that havebeen assigned a strain designation, and which have been characterizedfor short chain fatty acid (SCFA) production, antibiotic resistance,and/or analyzed by whole genome sequencing.

Table 6 below shows 25 examples of isolate samples, including theirgenus and species, which were characterized for short chain fatty acidproduction, antibiotic resistance, and/or by whole genome sequencing.

TABLE 6 Isolate Characterization Short Chain Fatty Acid Antibiotic WholeIsolate Production Resistance Genome Number Genus Species StrainDesignation Analysis Analysis Seq. 1 Akkermansia muciniphila A.muciniphila ST7 Y Y 2 Akkermansia muciniphila A. muciniphila ST6 Y 4Akkermansia muciniphila A. muciniphila ST5 Y 5 Akkermansia muciniphilaA. muciniphila ST1 (Am1) Y 11 Akkermansia muciniphila A. muciniphilaST15 (Am5) Y Y 18 Akkermansia muciniphila A. muciniphila ST22 (Am6) Y 28Akkermansia muciniphila A. muciniphila ST38 (Am4) Y 31 Akkermansiamuciniphila A. muciniphila ST43 (Am2) Y Y 33 Akkermansia muciniphila A.muciniphila ST46 (Am3) Y Y 39 Akkermansia muciniphila A. muciniphilaST58 (Am7) Y 111 Bifidobacterium adolescentis B. adolescentis ST15(Bs10) Y 113 Bifidobacterium longum B. longum ST27 (Bl9) Y 126 Blautiaproducta B. producta ST4 (Bp8) Y 147 Coprococcus comes C. comes ST3(Cc11) Y 184 Faecalibacterium prausnitzii F. prausnitzii ST23 (Fp13) Y Y188 Faecalibacterium prausnitzii F. prausnitzii ST27 (Fp14) Y Y 192Faecalibacterium prausnitzii F. prausnitzii ST38 (Fp12) Y Y Y 195Faecalibacterium prausnitzii F. prausnitzii ST58 (Fp15) Y Y 199Faecalibacterium prausnitzii F. prausnitzii ST74 (Fp16) Y Y 211Lachnoclostridium symbiosum L. symbiosum ST40 (Bp7) Y 214 Lactobacilluscrispatus L. crispatus ST100 (Lj20) Y Y 221 Lactobacillus jensenii L.jensenii ST10 (Lc18) Y 223 Lactobacillus johnsonii L. johnsonii ST8(Lg19) Y 225 Lactobacillus johnsonii L. johnsonii ST74 (Lj 17) Y 247Ruminococcus bromii R. bromii ST42 (Rb21) Y

Example 6 Species Listing for All Isolate Samples (Strains) 1-251

This example lists the isolates described herein by their species, andfurther provides information regarding their strain, phylum, and genus.As mentioned and described above, the isolates belong to the kingdom ofbacteria.

Table 7 below shows that the 251 isolated bacterial colonies belong to64 different species. Municiphila is the most abundant species among allisolated colonies with 36 total isolate numbers. Additional species withisolate numbers >10 are prausnitzii, fergusonii, vulgatus, and fragilis.

TABLE 7 Species Listing for All Isolate Samples (Strains) 1-251 Numberof Number Strain Isolates Phylum Genus Species 1 Akkermansia muciniphila36 Verrucomicrobia Akkermansia muciniphila 2 Anaerostipes hadrus 1Firmicutes Anaerostipes hadrus 3 Anaerotignum lactatifermentans 1Firmicutes Anaerotignum lactatifermentans 4 Bacteroides caccae 2Bacteroidetes Bacteroides caccae 5 Bacteroides dorei 3 BacteroidetesBacteroides dorei 6 Bacteroides faecis 9 Bacteroidetes Bacteroidesfaecis 7 Bacteroides finegoldii 2 Bacteroidetes Bacteroides finegoldii 8Bacteroides fragilis 14 Bacteroidetes Bacteroides fragilis 9 Bacteroidesintestinalis 1 Bacteroidetes Bacteroides intestinalis 10 Bacteroidesstercoris 8 Bacteroidetes Bacteroides stercoris 11 Bacteroidesthetaiotaomicron 10 Bacteroidetes Bacteroides thetaiotaomicron 12Bacteroides uniformis 3 Bacteroidetes Bacteroides uniformis 13Bacteroides vulgatus 14 Bacteroidetes Bacteroides vulgatus 14Bacteroides xylanisolvens 2 Bacteroidetes Bacteroides xylanisolvens 15Bifidobacterium faecale 2 Actinobacteria Bifidobacterium faecale 16Bifidobacterium longum 1 Actinobacteria Bifidobacterium longum 17Bifidobacterium stercoris 2 Actinobacteria Bifidobacterium stercoris 18Blautia faecis 2 Firmicutes Blautia faecis 19 Blautia gnavus 1Firmicutes Blautia gnavus 20 Blautia luti 1 Firmicutes Blautia luti 21Blautia obeum 6 Firmicutes Blautia obeum 22 Blautia producta 10Firmicutes Blautia producta 23 Blautia stercoris 2 Firmicutes Blautiastercoris 24 Blautia torques 1 Firmicutes Blautia torques 25 Blautiawexlerae 7 Firmicutes Blautia wexlerae 26 Collinsella aerofaciens 1Actinobacteria Collinsella aerofaciens 27 Coprococcus comes 8 FirmicutesCoprococcus comes 28 Coprococcus eutactus 1 Firmicutes Coprococcuseutactus 29 Dorea formicigenerans 3 Firmicutes Dorea formicigenerans 30Dorea longicatena 1 Firmicutes Dorea longicatena 31 Eisenbergiellamassiliensis 1 Firmicutes Eisenbergiella massiliensis 32Erysipelatoclostridium ramosum 7 Firmicutes Erysipelatoclostridiumramosum 33 Escherichia fergusonii 13 Proteobacteria Escherichiafergusonii 34 Eubacterium hallii 1 Firmicutes Eubacterium hallii 35Eubacterium ventriosum 1 Firmicutes Eubacterium ventriosum 36Faecalibacterium prausnitzii 16 Firmicutes Faecalibacterium prausnitzii37 Holdemanella biforme 3 Firmicutes Holdemanella biforme 38 Hungatellaeffluvii 1 Firmicutes Hungatella effluvii 39 Hungatella hathewayi 1Firmicutes Hungatella hathewayi 40 Lachnoclostridium aerotolerans 2Firmicutes Lachnoclostridium aerotolerans 41 Lachnoclostridium aldenense1 Firmicutes Lachnoclostridium aldenense 42 Lachnoclostridiumasparagiforme 1 Firmicutes Lachnoclostridium asparagiforme 43Lachnoclostridium bolteae 1 Firmicutes Lachnoclostridium bolteae 44Lachnoclostridium lavalense 1 Firmicutes Lachnoclostridium lavalense 45Lachnoclostridium symbiosum 1 Firmicutes Lachnoclostridium symbiosum 46Lachnospira pectinoschiza 1 Firmicutes Lachnospira pectinoschiza 47Lactobacillus coleohominis 1 Firmicutes Lactobacillus coleohominis 48Lactobacillus crispatus 3 Firmicutes Lactobacillus crispatus 49Lactobacillus gasseri 1 Firmicutes Lactobacillus gasseri 50Lactobacillus jensenii 5 Firmicutes Lactobacillus jensenii 51Lactobacillus johnsonii 4 Firmicutes Lactobacillus johnsonii 52Lactonifactor longoviformis 1 Firmicutes Lactonifactor longoviformis 53Longibaculum muris 4 Firmicutes Longibaculum muris 54 Muribaculumintestinale 1 Bacteroidetes Muribaculum intestinale 55 Oscillibacterruminantium 4 Firmicutes Oscillibacter ruminantium 56 Parabacteroidesdistasonis 1 Bacteroidetes Parabacteroides distasonis 57 Parabacteroidesmerdae 5 Bacteroidetes Parabacteroides merdae 58 Propionibacterium acnes1 Actinobacteria Cutibacterium acnes 59 Roseburia inulinivorans 3Firmicutes roseburia inulinivorans 60 Ruminococcus bromii 1 FirmicutesRuminococcus bromii 61 Shigella flexneri 1 Proteobacteria Shigellaflexneri 62 Staphylococcus epidermidis 1 Firmicutes Staphylococcusepidermidis 63 Staphylococcus warneri 1 Firmicutes Staphylococcuswarneri 64 Sutterella wadsworthensis 1 Proteobacteria Sutterellawadsworthensis

Example 7 The Microbial Consortium TC3 Reduces Immune Activation inMouse Model

This example shows that the three-member microbial consortium (e.g.,TC3) consisting of Lactobacillus johnsonii, Faecalibacteriumprausnitzii, and Akkermansia muciniphila effectively reduced immuneactivation demonstrated in an allergic sensitization mouse model and asdepicted by FIG. 1A-FIG. 1L. The bacterial strains Lactobacillusjohnsonii, Faecalibacterium prausnitzii, and Akkermansia muciniphila ofthe TC3 microbial consortium used for this study were provided by theAmerican Type Culture Collection (ATCC).

In order to investigate the protective effects of the oral TC3supplementation the cockroach allergen (CRA) murine model was used.C57BL/6 mice (7-8 weeks old) were intratracheally sensitized (Day 1-3)and subsequently challenged for one week with cockroach allergen (CRAand TC3 cohorts only). The mice of the CRA and TC3 cohorts wereconcurrently supplemented with phosphate buffered saline (PBS, negativevehicle control for CRA cohorts) or with the microbial consortium TC3(TC3 cohort). In the first week supplementation was performed daily,followed by supplementation twice a week for the remaining two weeks.All supplementations were performed by oral gavage using bacteriaresuspended in 100 μl of PBS. At the conclusion of the study, mice wereeuthanized, and various tissues (lung, spleen, ileum) were collected forex vivo analyses.

Three independent studies in non-treated (NT) mice, cockroach allergen(CRA) treated mice, and mice treated with CRA and an oralsupplementation containing a microbial consortium (TC3) were performed.The plasma concentration values (as ng/mL or pg/mL) of IgE, CRA-specificIgE and histamine were then measured in all cohorts (FIG. 1A-FIG. 1C)using ELISA. Further, the absolute concentration values (in pg/mL, FIG.1D-FIG. 1E) and the fold change (FIG. 1G-FIG. 1H) of the inflammatorycytokines IL-4 and IL-13 in lung tissue were measured by performingELISA and qPCR, respectively.

In addition, the abundance of regulatory T cells (FIG. 1I), type 2helper T cells (FIG. 1J), eosinophils (FIG. 1K), and neutrophils (FIG.1L) in lung tissues in all three study arms was measured using flowcytometry (fluorescence-assisted cell sorting).

Using standard t-test, significant reductions in circulating IgE andinflammatory cytokines were observed in cohorts treated with themicrobial consortium TC3 when compared to cohorts that were exposed toonly CRA. Further, oral supplementation with the TC3 consortiumincreased the amount of anti-inflammatory, regulatory T cells, andreduced the amount of inflammatory type 2 helper T cell populations inlung tissue.

Example 8 The Use of Isolates 1-251 in an Allergic Sensitization MouseModel

This example shows the use of any of the isolated bacteria strains 1-251listed in Table 3 for oral supplementation to effectively reduce theimmune activation in an allergic sensitization mouse model.

The cockroach allergen (CRA) murine model as described above (EXAMPLE 6)is used to investigate the protective effects of the isolates 1-251listed in Table 3 in a microbial consortium administered as an oralsupplementation.

The isolated bacterial strains Lactobacillus johnsonii, Faecalibacteriumprausnitzii, and Akkermansia muciniphila from Table 3 (labeled withwild-type, WT, in FIG. 2A-FIG. 2U) show the same reduction in immuneactivation in the allergic sensitization (CRA) murine model whencompared to strains that are provided by the vendor ATCC (FIG. 2A-FIG.2U).

Example 9 Determination of Strain Properties and Characteristics for A.muciniphila ST7 (AM-ST7)

This example shows the determination of strain properties andcharacteristics for A. muciniphila ST7 (or AM-ST7).

Akkermansia muciniphila ST7 used in this example was isolated from thestool of a healthy human.

Whole Genome Sequencing and Comparative Analysis

The genome of Akkermansia muciniphila ST7 was sequenced and analyzed. Toachieve this, DNA was extracted from AM-ST7 and next-generation DNAsequencing and genome assembly was performed. The AM-ST7 genome librarywas sequenced using an Ion-Torrent Sequencer, sequence data wasassembled using the SPAdes Assembler, and reads were aligned to thereference genome using Harvest. The AM-ST7 genome was sequenced to anaverage depth of 469X coverage, allowing for high confidence SNP callsfor comparison with the type strain A. muciniphila BAA-835. To that end,genome scaffolds were multiply-aligned with a fast core-genomemulti-aligner (Parsnp v1.2) against the reference of the representativebacterial genome (reference Akkermansia muciniphila ATCC BAA-835). Thealignment was then used to create phylogenetic trees to compare andidentify the closest related strains to the bacterial genome of AM-ST7using clade clusters (FIG. 3 ). As expected due to the conserved natureof A. muciniphila, AM-ST7 clusters closely with BAA-835.

A comparative genomic analysis between AM-ST7 and the type strain A.muciniphila BAA-835 was also performed (FIG. 4 -FIG. 5 ). The A.muciniphila ST7 genome shares 94% nucleotide identity with A.muciniphila BAA-835, with 2,751,252 and 2,664,102 total nucleotides and55.5% and 55.8% GC content, respectively. The genomic analysis revealed220 vs. 218 subsystems and 2,815 vs. 2,577 coding regions in A.muciniphila ST7 and A. muciniphila BAA-835, respectively. RapidAnnotation Subsystem Technology (RAST) was leveraged for predictivefunctional analysis and gene annotation. This analysis showed that theA. muciniphila ST7 genome contains the same major functional categoriesas A. muciniphila BAA-835, with a small number of differing genes.Without being bound by any theory, these difference may correspond withfunctional (e.g., therapeutic) differences specific to AM-ST7 (FIG. 6 ).

Antibiotic Resistance, Virulence Factors, and Viruses

In addition to providing high-resolution microbial identification, highperformance data mining algorithms and highly curated dynamic comparatordatabases (e.g., GenBook®) were used to inspect the genome for thepresence of virulence, antibiotic resistance, and viral geneticcomponents. GenBook® is a highly curated, proprietary genomic database,comprising 150,000+bacteria, viruses, fungi and protists genomes andgene sequences. To process the sequence data, a high-performance datamining K-mer based algorithm that rapidly disambiguates hundreds ofmillions of short reads of a metagenomic sample into the discretemicroorganisms engendering the particular sequences was used. Thepipeline had two separable comparators. The first consisted of apre-computation phase and a per-sample computation. The input to thepre-computation phase was a reference microbial database, and its outputwas a whole genome phylogeny tree, together with sets of fixed lengthk-mer fingerprints (biomarkers) that were uniquely identified withdistinct nodes of the tree. The second per-sample, computational phasesearched the hundreds of millions of short sequence reads against thefingerprint sets in minutes. The resulting statistics were analyzed togive fine-grain composition and relative abundance estimates at allnodes of the tree. Enhanced discriminatory power was achieved by runningthe comparators in sequence. The first comparator found reads in whichthere is an exact match with an n-mer uniquely identified with a set ofreference strains; the second comparator then statistically scored theentire read against the reference to verify that the read was indeeduniquely identified with that set. Similarly, the sample resistome andvirulome, the collection of antibiotic resistance and virulence genes inthe sample, was identified using the CosmosID, Inc. bioinformaticssoftware package to query the unassembled sequence reads against theCosmosID curated antibiotic resistance and virulence gene databases.This analysis of the AM-ST7 genome found that it does not harborantibiotic resistance cassettes or pathogenicity islands (virulencefactors). Additionally, this analysis found that AM-ST7 does not harbormammalian viruses.

Antibiotic susceptibility was then determined by standard Kirby-Bauerdisk diffusion testing. AM-ST7 was susceptible to the followingantibiotics (cm=zone of inhibition): azithromycin (2.5 cm),amoxicillin/clavulanic acid (4 cm), vancomycin (1.5 cm), tetracycline (3cm), bacitracin (2 cm), and levofloxacin (1.2 cm).

Batch Characterization

All AM-ST7 drug substance batches produced underwent the followingcharacterization to confirm identity.

Full-Length 16S rRNA Gene Sequencing. The identity of each batch ofAM-ST7 cells was confirmed using the genotypic microbial identificationservice including the MicroSEQ Rapid Microbial Identification System(Applied Biosystems) for genotypic identification of unknown bacterialsamples using 16S ribosomal RNA gene sequencing (FIG. 7 ). The 16Sribosomal RNA gene has been used to identify bacterial isolates, andthis method amplifies, sequences, and analyzes the full 1,500 base pairsof the 16S rRNA gene, including several hypervariable regions withunique DNA sequences that can be used to confirm the identification ofbacterial species and determine phylogenetic relatedness of bacterialsamples. To acquire the species level identification of, the sample wasprocessed according internal standard operating procedures, and theresulting quality control values, bacterial species identification, andpercent identity scores.

Macroscopic Colony Morphology Analysis. The identification of the cellsin each AM-ST7 drug substance batch was evaluated using standardoperating procedures for microbiological evaluations of A. muciniphilaunder cGMP. This procedure includes macroscopic colony morphologyanalysis of the drug substance. Briefly, a representative aliquot of thedrug substance was cultured on mucin agar under anaerobic conditions.The colony morphology of the resulting cells was visually evaluated todetermine if the cells fall within known specifications for AM-ST7colony morphology. The colony morphology characteristics evaluatedinclude colony shape, colony margin (edge), colony elevation, colonysize, colony texture, appearance, pigmentation, and optical property.

Microscopic Cell Morphology Analysis. The identification of the cells ineach AM-ST7 drug substance batch was evaluated using standard operatingprocedure for microbiological evaluations of A. muciniphila under cGMP.This procedure included microscopic cell morphology analysis of the drugsubstance. To perform microscopic analysis of the cell morphologiespresent in each culture, a 10 μL loop of concentrated cell stock wasstreaked onto a glass slide. The cells were then heat fixed and Gramstained using standard procedures. After Gram staining, a coverslip wasadded and the slide was viewed using a 1000×oil immersion objective.Slides were inspected to determine if the drug substance cells meetknown cell morphology and gram-stain characteristics of AM-ST7.

This data demonstrates various properties of the strain Akkermansiamuciniphila ST7 (AM-ST7) that can be used in a therapeutic compositionas described herein for the prevention and treatment of diseases andconditions (e.g., dysbiosis, inflammation, etc.).

Example 10

Determination of Strain Properties and Characteristics forFaecalibacterium prausnitzii ST38 (or FP-ST38)

This example shows the determination of strain properties andcharacteristics for Faecalibacterium prausnitzii ST38 (or FP-ST38).Faecalibacterium prausnitzii ST38 (FP-ST38) used in this example wasisolated from the stool of a healthy human.

Whole Genome Sequencing and Comparative Analysis

The genome of Faecalibacterium prausnitzii ST38 has been sequenced andanalyzed as described above in EXAMPLE 8. The multi-alignment was usedto create phylogenetic trees to compare and identify the closest relatedstrains to our bacterial genome using clade clusters. Notably, FP-ST38clusters in a distinct clade from that of the reference strain (FIG. 8-FIG. 9).

Antibiotic Resistance, Virulence Factors, and Viruses

The genome of this strain was inspected for the presence of virulence,antibiotic resistance, and viral genetic components as described abovein EXAMPLE 8. The analysis of the FP-ST38 genome found that it does notharbor antibiotic resistance cassettes or pathogenicity islands(virulence factors). Additionally, the above analysis found that FP-ST38does not harbor mammalian viruses.

Antibiotic susceptibility was determined by standard Kirby-Bauer diskdiffusion testing. FP-ST38 is susceptible to the following antibiotics(cm=zone of inhibition): amoxicillin/clavulanic acid (2.5 cm),ceftriaxone (2 cm), tetracycline (5 cm), amikacin (2 cm), and bacitracin(3.4 cm).

Batch Characterization

All FP-ST38 drug substance batches produced underwent the samecharacterization procedure as described above in EXAMPLE 8.

Generally, batch characterization was conducted as described above inEXAMPLE 8.

This data demonstrates various properties of the strain Faecalibacteriumprausnitzii ST38 (or FP-ST38) that can be used in a therapeuticcomposition as described herein for the prevention and treatment ofdiseases and conditions (e.g., dysbiosis, inflammation, etc.).

Example 11

Determination of Strain Properties and Characteristics for Lactobacilluscrispatus ST100 (LC-ST-100)

This example shows the determination of strain properties andcharacteristics for Lactobacillus crispatus ST100 (LC-ST100).Lactobacillus crispatus ST100 (LC-ST100) was isolated from a vaginalswab from a healthy human adult female.

Whole Genome Sequencing and Comparative Analysis

The genome of Lactobacillus crispatus ST100 (LC-ST100) has beensequenced and analyzed as described above in EXAMPLE 8. Themulti-alignment was used to create phylogenetic trees to compare andidentify the closest related strains to our bacterial genome using cladeclusters. Notably, LC-ST100 clusters in a distinct clade from that ofthe reference strain (FIG. 10 -FIG. 11 ).

Comparative analysis of the LC-ST100 genome to the related strain L.crispatus CTV-05, which has been safely used as a vaginal suppository inhuman clinical trials, was also performed. The genome of LC-ST100 shares78% nucleotide identity with CTV-05, revealing similar genome sizes(2,465,006 and 2,299,477 respectively) and GC content (38.8% and 37.1%,respectively). Comparison of the predictive functional capacity for bothstrains using RAST—Rapid Annotation Subsystem Technology suggestssignificant overlap between the two strains with 96-88% overlap at thecategory, subcategory, and subsytems levels. Interestingly, LC-ST100harbors additional genes related to nitrogen metabolism and aromaticcompound metabolism when compared to strain CTV-05. The sponsor iscontinuing in their efforts to characterize the unique genomicattributes of LC-ST100, in addition to metabolic and phenotypicdifferences.

Antibiotic Resistance, Virulence Factors, and Viruses

The genome of this strain was inspected for the presence of virulence,antibiotic resistance, and viral genetic components as described abovein EXAMPLE 8. The above analysis of the LC-ST100 genome found that itdoes not harbor antibiotic resistance cassettes or pathogenicity islands(virulence factors). Additionally, the above analysis found thatLC-ST100 does not harbor mammalian viruses.

Antibiotic susceptibility was determined by standard Kirby-Bauer diskdiffusion testing. LC-ST100 is susceptible to the following antibiotics(cm =zone of inhibition): azithromycin (3.5 cm), amoxicillin/clavulanicacid (5 cm), clindamycin (4 cm), vancomycin (3.8 cm), ceftriaxone (3.5cm), tetracycline (4.2 cm), ampicillin (4 cm), bacitracin (2.5 cm).

Batch Characterization

All LC-ST100 drug substance batches produced underwent the samecharacterization procedure as described above in EXAMPLE 8.

Generally, batch characterization was conducted as described above inEXAMPLE 8.

This data demonstrates various properties of the strain Lactobacilluscrispatus ST100 (LC-ST100) that can be used in a therapeutic compositionas described herein for the prevention and treatment of diseases andconditions (e.g., dysbiosis, inflammation, etc.).

Example 12 Chemical Analysis of the three strains A. muciniphila ST7,Faecalibacterium prausnitzii ST38, and Lactobacillus crispatus ST100

This example shows the chemical and metabolic analysis of the strains A.muciniphila ST7, Faecalibacterium prausnitzii ST38, and Lactobacilluscrispatus ST100. First, chemical profiles of the spent media and cellsof three strains A. muciniphila ST7, Faecalibacterium prausnitzii ST38,and Lactobacillus crispatus ST100 in comparison to their respectivenaïve media were analyzed. Samples were analyzed using liquidchromatography coupled with mass spectrometry (LC/MS) profiling with afocus on mid-polar compounds. Second, the quantitation of small chainfatty acids (SCFA) in these samples was conducted using gaschromatography coupled with mass spectrometry (GC/MS). Third, thebiological activity of these three strains against Candida sp. andRhodotorula sp. was tested in-vitro.

Materials and Methods

Generally, all samples were provided in lyophilized from. Subsets ofeach sample were suspended in solvent for analysis by LC/MS and GC/MS.Following these analyses, further subsets of each sample were extractedand fractionated (to enrich the medium polar range). Fractions wereanalyzed by LC/MS. Cell extracts were analyzed in a bioassay againstCandida sp. and Rhodotorula sp. in a disk diffusion assay at 3concentrations.

Table 8 below described the samples tested in this example.

TABLE 8 Sample Descriptions Sample Description 1 Lactobacillus crispatuslyophilized cells 2 Lactobacillus crispatus lyophilized spent media 3Lactobacillus crispatus lyophilized naïve media 4 Akkermansiamuciniphila lyophilized cells 5 Akkermansia muciniphila lyophilizedspent media 6 Akkermansia muciniphila lyophilized naïve media 7Faecalibacterium prausnitzii lyophilized cells 8 Faecalibacteriumprausnitzii lyophilized spent media 9 Faecalibacterium prausnitziilyophilized naïve media

Analytical Preparation and Direct Profiling. Samples for LC/MS profilingwere generated by transferring 30 mg of the sample material into a 4 mLglass vial followed by addition of either 500 μL Methanol and 500 μLwater or 1 mL of DMSO. The samples were then sonicated for up to 10 minand non-dissolved particles were removed be centrifugation followed byfiltration.

Preparative Cell Extraction. Lyophilized cells were extracted prior tofractionation/bio assay to remove cell wall debris. The cells wereadjusted to a final concentration of 50 mg/mL (Table 9) and sonicatedfor 15 min followed by vortexing for 2 min and a second sonication stepof 15 min. Afterwards, 10 mL of the suspension were transferred toanother vial an centrifuged at 10,000 RCF for 5 min. The supernatant wastransferred to a new vial.

TABLE 9 Sample Amounts and Extraction Volumes Amount 90% Methanol Finalconcentration Sample [mg] (aq.) [mL] [mg/mL] 1 584 11.67 50 4 631 12.6250 7 610 12.2  50

Disk diffusion test. Disk diffusion tests for antimicrobial activitywere prepared according to DIN 58940-3 (DIN-Taschenbuch 222, 5. Edition)using the strains 00549fBCD000864 (Rho. Glutinis, test strain #1) DSM70821 and 00539fDSM011226 (Ca. glabrata, test strain #2) DSM 11226. Forthe in vitro testing of the biological activity for the threetherapeutic strains A. muciniphila ST7, Faecalibacterium prausnitziiST38, and Lactobacillus crispatus ST100, 73.4 mg Cycloheximid weredissolved in 7.34 mL 90% MeOH (aq.) solution resulting in a 10 mg/mLsolution. 3 pieces of aluminum foil (10×10 cm) were sterilized using UVlight for 30 min. Four sterile filterdiscs were placed on each piece offoil. The dilutions 1-3 (15 μL) were pipetted to the first threefilterdisc. The fourth filterdisc was prepared with 10 μl ofcycloheximid solution. All spots were allowed to dry completely.

The test strains were diluted to 1.000.000 cfu/mL using saline solution.Agar plates were prepared using modified YNB-Agar. The test strainsolution (400 μL) was pipetted to the center of the plate anddistributed homogenously using a sterile Grigalski-spatula. Afterapproximately 5 min the filterdiscs were placed on the plate which wasclosed with parafilm directly and incubated for 24 h at 25° C. After 24h the zone of inhibition was measured and recorded in Table 10 below.

TABLE 10 Disk Diffusion Assay Results Diameter Diameter μg/discinhibition inhibition Sample or μg zone* zone* Test (see V [μL]/extracted/ [mm] [mm] strain Table 6) disc disc Sample Pos. Control** 2 230 1500 6 11 2 5 30 1500 6 11 2 8 30 1500 6 11 2 9 30 1500 6 10 2 3 301500 6 10 2 6 30 1500 6 10 2 1 30 3000 6 9 2 4 30 3000 6 9 2 7 30 3000 69 2 2 10 500 6 11 2 5 10 500 6 11 2 8 10 500 6 11 2 9 10 500 6 10 2 3 10500 6 10 2 6 10 500 6 10 2 1 10 1000 6 9 2 4 10 1000 6 9 2 7 10 1000 6 91 2 30 1500 6 14 1 5 30 1500 6 14 1 8 30 1500 6 14 1 9 30 1500 6 13 1 330 1500 6 13 1 6 30 1500 6 13 1 1 30 3000 6 13 1 4 30 3000 6 13 1 7 303000 6 13 1 2 10 500 6 14 1 5 10 500 6 14 1 8 10 500 6 14 1 9 10 500 613 1 3 10 500 6 13 1 6 10 500 6 13 1 1 10 1000 6 13 1 4 10 1000 6 13 1 710 1000 6 13 *Disk diameter = 6 mm **Positive control = 2 μgCycloheximide/disc

Sample fractionation. Naive and spent media samples were fractionateddirectly using 2 g of sample. Approximately 500 mg of lyophilized cellswere extracted as described herein and fractionated. The material wasadsorbed on celite (14 g) and fractionated using a Polygoprep 60 μm125×15 mm column, generating 18 fractions.

Quantitation of SCFAs. Samples were dissolved in 1 mL of ultrapurewater, followed by centrifugation. The supernatants were collected andtransferred to a GC vial. For quality control, a mixed pooled sample (QCsample) was created by taking a small aliquot from each sample. Thissample was analyzed at regular intervals throughout the sequence. Matrixeffects were tested for quantified compounds by spiking the QC sample ina minimum of two levels. The small chain fatty acids (SCFAs) method is aGC-MS method specially designed for short-chain fatty acids using a highpolarity column. Samples were acidified with hydrochloric acid.

Tables 11-13 below show fractions generated from the three strains A.muciniphila ST7, Faecalibacterium prausnitzii ST38, and Lactobacilluscrispatus ST100, respectively.

TABLE 11 Fractions Generated from Lactobacillus crispatus spent amountnaïve amount cell amount medium [mg] medium [mg] extract [mg] C-3030-A925.0 C-3031-A 1789.5 C-3043-A 25.4 C-3030-B 1095.6 C-3031-B 603.4C-3043-B 12.0 C-3030-C 81.5 C-3031-C 61.4 C-3043-C 6.3 C-3030-D 38.2C-3031-D 35.5 C-3043-D 4.3 C-3030-E 83.9 C-3031-E 122.6 C-3043-E 8.1C-3030-F 66.8 C-3031-F 38.3 C-3043-F 3.6 C-3030-G 19.9 C-3031-G 14.2C-3043-G 3.5 C-3030-H 10.3 C-3031-H 8.1 C-3043-H 5.2 C-3030-I 6.0C-3031-I 4.4 C-3043-I 3.9 C-3030-K 4.7 C-3031-K 3.1 C-3043-K 3.3C-3030-L 5.7 C-3031-L 2.5 C-3043-L 5.8 C-3030-M 12.6 C-3031-M 1.9C-3043-M 5.1 C-3030-N 6.7 C-3031-N 1.7 C-3043-N 1.4 C-3030-O 4.4C-3031-O 1.5 C-3043-O 2.5 C-3030-P 2.6 C-3031-P 1.0 C-3043-P 1.7C-3030-R 1.2 C-3031-R 1.2 C-3043-R 3.8 C-3030-S 0.0 C-3031-S 1.0C-3043-S 0.9 C-3030-T 0.4 C-3031-T 0.2 C-3043-T 2.4

TABLE 12 Fractions Generated from Akkermansia muciniphila spent amountnaïve amount cell amount medium [mg] medium [mg] extract [mg] C-3032-A1060.7 C-3033-A 1390.8 C-3044-A 27.0 C-3032-B 375.1 C-3033-B 685.4C-3044-B 8.4 C-3032-C 58.8 C-3033-C 82.3 C-3044-C 3.9 C-3032-D 29.2C-3033-D 43.2 C-3044-D 3.1 C-3032-E 126.2 C-3033-E 152.2 C-3044-E 5.8C-3032-F 48.6 C-3033-F 33.4 C-3044-F 2.2 C-3032-G 23.8 C-3033-G 10.5C-3044-G 3.1 C-3032-H 5.9 C-3033-H 7.9 C-3044-H 1.8 C-3032-I 2.3C-3033-I 2.8 C-3044-I 1.9 C-3032-K 1.6 C-3033-K 2.0 C-3044-K 2.6C-3032-L 1.0 C-3033-L 1.8 C-3044-L 1.9 C-3032-M 1.5 C-3033-M 1.7C-3044-M 2.1 C-3032-N 0.7 C-3033-N 1.6 C-3044-N 2.3 C-3032-O 0.5C-3033-O 1.4 C-3044-O 3.1 C-3032-P 0.4 C-3033-P 1.0 C-3044-P 3.2C-3032-R 0.5 C-3033-R 0.7 C-3044-R 3.1 C-3032-S 0.4 C-3033-S 0.8C-3044-S 1.4 C-3032-T 0.2 C-3033-T 0.3 C-3044-T 0.5

TABLE 13 Fractions Generated from Faecalibacterium prausnitzii spentamount naïve amount cell amount medium [mg] medium [mg] extract [mg]C-3034-A 1316.8 C-3035-A 1529.2 C-3045-A 17.2 C-3034-B 830.2 C-3035-B579.1 C-3045-B 10.6 C-3034-C 90.0 C-3035-C 81.8 C-3045-C 3.2 C-3034-D48.9 C-3035-D 52.5 C-3045-D 4.6 C-3034-E 169.8 C-3035-E 175.3 C-3045-E3.8 C-3034-F 51.4 C-3035-F 35.7 C-3045-F 2.5 C-3034-G 14.5 C-3035-G 11.7C-3045-G 0.9 C-3034-H 7.3 C-3035-H 5.9 C-3045-H 1.3 C-3034-I 3.4C-3035-I 1.8 C-3045-I 1.5 C-3034-K 2.6 C-3035-K 0.8 C-3045-K 1.0C-3034-L 2.4 C-3035-L 0.7 C-3045-L 1.6 C-3034-M 2.2 C-3035-M 2.0C-3045-M 3.8 C-3034-N 1.5 C-3035-N 1.9 C-3045-N 3.5 C-3034-O 1.9C-3035-O 1.4 C-3045-O 6.1 C-3034-P 0.3 C-3035-P 1.2 C-3045-P 1.8C-3034-R 1.3 C-3035-R 1.0 C-3045-R 0.5 C-3034-S 0.4 C-3035-S 0.8C-3045-S 0.9 C-3034-T 0.4 C-3035-T 0.6 C-3045-T 0.6

LC/MS Analysis Results

LC/MS profiling of the analytical preparations of 9 samples showed nosignificant signals in the medium polar range (approx. 5-30 min.). Tofurther address this finding, samples were subjected to a preparativeextraction and RP-HPLC fractionation to see if any enrichment of themedium polar range may be achieved (Tables 11-13). After subtracting theprofile of the naive medium, one fraction, fraction C-3044M, derivedfrom Akkermansia muciniphila cells, showed significant peaks of interest(FIG. 12 ).

The MS data suggested these compounds to be phosphatidylcholineprecursors and/or phosphatidylcholine-like compounds (compounds 1-3)with chemical structures shown in FIG. 13 . This data confirms thatAkkermansia muciniphila produces significant quantities ofanti-inflammatory compounds, including phosphatidylcholines andderivatives thereof, resulting, at least in part, in the therapeuticproperties of Akkermansia muciniphila-containing microbial consortiadescribed herein.

GC/MS Analysis Results

GC/MS analysis of the samples screened for ten SCFAs as described belowin Table 14. Acetic acid was detected in all samples. Formic acid wasdetected in A. muciniphila and F. prausnitzii. Propanoic acid wasdetected in F. prausnitzii, and 2-methyl-propanoic acid was detected inL. crispatus. Butanoic acid was detected in the cells and spent media ofF. prausnitzii (and not the naive media) confirming that F. prausnitziiproduces butyrate.

TABLE 14 Quantification of SCFAs* 2-methyl- 3-methyl- 4-methyl- AceticFormic Propanoic propanoic Butanoic butanoic Pentanoic pentanoicHexanoic Heptanoic acid acid acid acid acid acid acid acid acid acidDescriptive 38.0 52.4 52.6 3.3 30.5 ND ND ND ND ND power Rel. 2 4 2 8  6 ND ND ND ND ND Precision (%) Absolute 8 4 0.5 0.1 4 ND ND ND ND NDPrecision LOD 4 5 1.3 0.4 3 0.3 0.4 0.5 0.3 0.6 Sample μmol/g μmol/gμmol/g μmol/g μmol/g μmol/g μmol/g μmol/g μmol/g μmol/g 1 125 <LOD <LOD<LOD <LOD <LOD <LOD <LOD <LOD <LOD 1 113 <LOD <LOD <LOD <LOD <LOD <LOD<LOD <LOD <LOD 2 523 <LOD <LOD 0.7 <LOD <LOD <LOD <LOD <LOD <LOD 2 499<LOD 1.4 0.5 4.3 <LOD <LOD <LOD <LOD <LOD 3 1029 6 <LOD 1.5 <LOD <LOD<LOD <LOD <LOD <LOD 4 26 <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD 427 <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD <LOD 5 181 10 <LOD <LOD <LOD<LOD <LOD <LOD <LOD <LOD 5 174 10 <LOD <LOD <LOD <LOD <LOD <LOD <LOD<LOD 6 57 7 <LOD 0.5 <LOD <LOD <LOD <LOD <LOD <LOD 7 98 52 13 <LOD 100<LOD <LOD <LOD <LOD <LOD 7 86 36 9.4 <LOD 68 <LOD <LOD <LOD <LOD <LOD 8678 571 76 <LOD 369 <LOD <LOD <LOD <LOD <LOD 8 685 537 75 0.5 355 <LOD<LOD <LOD <LOD <LOD 9 803 13 45 0.8 <LOD <LOD <LOD <LOD <LOD <LOD *Cellsand spent media were analyzed in duplicate and compared to the naïvemedium. Naïve media is denoted by grey highlighting. The DescriptivePower is calculated as the ratio between the standard deviation withinexperimental samples and the standard deviation within the QC samples.Variables with a ratio higher than 2.5 are most likely to describevariation related to the experimental design. <LOD denotes thatmeasurement was below limit of detection.

Antimicrobial Testing Results

The screening of naive media, spent media, and cell extracts of allthree strains against Ca. glabrata and Rho. glutinis resulted in noobserved anti-microbial activity against these strains (Table A2).

The data of this study demonstrate that the herein analyzed samplesobtained from bacteria of microbial consortia primarily comprised ofcompounds in the polar region. Fractionation of the samples allowed foran analysis of the medium polarity range independently. The data furthershow that three phosphatidylcholine derivatives (compounds 1-3, FIG. 13) were identified as products of Akkermansia muciniphila. An additionalcompound with a molecular weight of 386 Da was also detected. Analysisof the small chin fatty acid (SCFA) content confirmed that F.prausnitzii produces butanoic acid, while acetic acid, formic acid,propanoic acid, and 2-methyl-propanoic acid, may be detecteddifferentially across the three strains. Additionally, a test forantimicrobial activity against Candida sp. and Rhodotorula sp. wasnegative.

Particularly the production of the three phosphatidylcholine derivatives1-3 (FIG. 13 ) is an indication that Akkermansia muciniphila (and otherstrains of the consortia described herein) produces significant amountsof compounds with biological activity, including anti-inflammatoryactivity, and thus may, at least in part, explain the therapeuticproperties of the microbial consortia of the present disclosure.

Example 13 Analysis of Antibiotic Resistance of Akkermansia muciniphilaand Faecalibacterium prausnitzii Strains

This example shows the analysis of antibiotic resistance of the strainsA. muciniphila ST7, F. prausnitzii ST38, or L. crispatus ST100.Antibiotic resistance in this example was determined using the brothsolution method.

Antibiotic resistance in this example was determined using the brothsolution method. This procedure involved preparing two-fold dilutions ofantibiotics (e.g., 1, 2, 4, 8, and 16 μg/mL) in a liquid growth mediumdispensed in test tubes. The antibiotic-containing tubes were inoculatedwith a standardized bacterial suspension of about 1-5×10⁵ CFU/mL.Following overnight incubation at 37° C., the tubes were examined forvisible bacterial growth as evidenced by turbidity. The lowestconcentration of antibiotic that prevented growth represented theminimal inhibitory concentration (MIC).

FIG. 14 shows the antibiotics tested and the results obtained fromantibiotic resistance experiment. A. muciniphila ST7 appeared to besusceptible to azithromycin, clinadmycin, and tetracycline. F.prausnitzii ST38 appeared to be susceptible to amikacin, azithromycin,clinadmycin, tetracycline, and vacomycin. L. crispatus ST100 appeared tobe susceptible to ampicillin, azithromycin, clinadmycin, tetracycline,and vacomycin.

Example 14 Analysis of Short-Chain Fatty Acid Production in CultureSupernatants of Akkermansia muciniphila and Faecalibacterium prausnitziiStrains

This example shows the analysis of short-chain fatty acid (SCFA)production in brain heart infusion (BHI) culture and chopped meatcarbohydrate (CMC) culture supernatants of Akkermansia muciniphila (FIG.15A) and Faecalibacterium prausnitzii strains (FIG. 15B-FIG. 15D),respectively.

Quantification of short chain fatty acids in culture media was performedby liquid chromatograph-mass spectrometry (LC-MS/MS). Culture mediasamples were spiked with stable labelled internal standards followed byprotein precipitation with an organic solvent. After centrifugation topellet protein precipitate, the supernatant was derivatized. Thereaction mixture was diluted, and an aliquot was injected onto an LC-MSsystem comprising an Agilent 1290/AB Sciex QTrap 5500 LC MS/MS systemequipped with a C18 reversed phase UHPLC column. The mass spectrometerwas operated in negative mode using electrospray ionization (ESI). Thepeak area of the target analyte ions was measured against the area ofthe corresponding internal standard ion peaks. Quantitation wasperformed using a weighted linear least squares regression analysisgenerated from calibration standards prepared prior to each run.LC-MS/MS raw data were collected and processed using AB SCIEX softwareAnalyst 1.6.2. Data reduction and analysis was performed using MicrosoftExcel 2016, and the results of this study are shown in FIG. 15A-FIG.15C.

FIG. 15A shows that the A. muciniphila strains AM 2, AM 3, AM 5, AM 6,and AM 7 produced comparable amounts of acetate than the ATCC referencestrain, with AM 3 and AM 7 on the lower end.

FIG. 15B shows that the F. prausnitzii strains FP 12, FP 13, FP 14, FP15, and FP 16 produced higher amounts of acetate than the ATCC referencestrain. Surprisingly, the strains FP 15 and FP 16 produced more thantwice the amount of acetate compared to the ATCC reference strain.

FIG. 15C shows that the F. prausnitzii strains FP 12, FP 13, FP 14, FP15, and FP 16 produce significantly higher amounts of isobutyratecompared to the ATCC reference strain or the CMC media control.

These findings demonstrate that the A. muciniphila and F. prausnitziiisolates described herein can be superior at the production of shortchain fatty acids such as acetate and isobutyrate compared toconventional strains of these species.

Example 15 Therapeutic Consortia Produce Beneficial Metabolites

This example demonstrates that the therapeutic consortia of the presentdisclosure produce various metabolites (e.g., fatty acids such as SCFAs,lipids such as phospholipids, etc.) that can have beneficial (e.g.,therapeutic and/or preventative) properties.

The supernatant of a cell culture comprising a consortium comprising thestrains A. muciniphila ST7, F. prausnitzii ST38 and L. crispatus ST100is analyzed for metabolites as described above in EXAMPLE 12 and EXAMPLE14. Analysis of the supernatant shows that the consortium producessignificant amounts of fatty acids and lipids (e.g., phospholipids,including phosphatidylcholines and derivatives thereof).

The consortium comprising the strains A. muciniphila ST7, F. prausnitziiST38 and L. crispatus ST100 is administered to animals suffering from aninflammatory disease induced in a cockroach allergen (CRA) murine model.The mouse model was used as described in EXAMPLE 6. The animalsreceiving the therapeutic consortium show significantly reducedinflammation compared to control animals. Analysis of inflammatorymarkers shows a reduction in pro-inflammatory markers (e.g., IL-4,IL-13, T_(H)2 cells, etc.).

Ex vivo analysis of tissue samples (e.g., blood samples) demonstrate acorrelation between the production of beneficial metabolites by thetherapeutic consortium and the reduction in inflammation. This datademonstrates that a production of specific molecules such as fattyacids, lipids, and/or phospholipids by a therapeutic consortium asdescribed herein can reduce inflammation in a subject.

Example 16 Akkermansia muciniphila Produces Beneficial Metabolites

This example demonstrates that Akkermansia muciniphila (e.g., the strainA. muciniphila ST7) produces various metabolites (e.g., fatty acids suchas SCFAs, lipids such as phospholipids, etc.) that can have beneficial(e.g., therapeutic and/or preventative) properties.

The supernatant of a cell culture comprising Akkermansia muciniphila isanalyzed for metabolites as described above in EXAMPLE 12 and EXAMPLE14. Analysis of the supernatant shows that Akkermansia muciniphilaproduces significant amounts of fatty acids and lipids. Particularly,the results show that Akkermansia muciniphila produces certainphospholipids including phosphatidylcholine and derivatives thereof(e.g., compounds 1-3 shown in FIG. 13 ).

A pharmaceutical composition comprising a consortium comprisingAkkermansia muciniphila is administered to animals suffering from aninflammatory disease induced in a cockroach allergen (CRA) murine model.The mouse model was used as described in EXAMPLE 6. The animalsreceiving the therapeutic consortium show significantly reducedinflammation compared to control animals. Analysis of tissue samples(e.g., blood samples) show a reduction in inflammation can be correlatedwith the production of phosphatidylcholine and/or phosphatidylcholinederivatives by Akkermansia muciniphila. A control cohort that receives apharmaceutical composition without Akkermansia muciniphila showssignificantly reduced amount of phosphatidylcholine and/orphosphatidylcholine derivativeand higher levels of inflammation.

This data demonstrates that phosphatidylcholine and/orphosphatidylcholine derivative production of Akkermansia muciniphilastrains can result in a reduction in inflammation, suggesting thatspecific metabolic activities of microbial consortia can havetherapeutic and/or preventative effects in subjects, e.g., to reduceand/or prevent dysbiosis and/or inflammation.

Example 17 Consortia Producing Phosphatidylcholine-like CompoundsProtect Against Allergic Airway Inflammation

This example demonstrates that therapeutic microbial consortiacomprising Akkermansia muciniphila (e.g., the strain A. muciniphila ST7)produce phosphatidylcholines and derivatives thereof and protect againstallergic airway inflammation in a Cockroach allergen (CRA) sensitizationmodel.

FIG. 16 illustrates an experimental outline that is used to investigatethe beneficial, anti-inflammatory properties of the herein describedmicrobial consortia that can produce phosphatidylcholine-like compounds.This 28-day experimental procedure includes 4 cohorts of C57BL/6 mice(7-8 weeks old) that are intratracheally sensitized at days 8, 9, and10, and subsequently challenged with Cockroach allergen (CRA) at days 21and 28 of the study. The 4 cohorts (cohorts A-D) are being administeredon days 1, 3, 5, 7, 9, 11, and 13 by oral gavage with a suspensioncomprising either A) the AM-ST7 strain; B) a bacterial consortiumcomprising AM-ST7, FP-ST38, and LC-ST100, wherein different Akkermansiamuciniphila (AM) strains can be used, each producing different amounts(e.g., with different rates) of phosphatidylcholine-like compounds(e.g., those depicted in FIG. 13 ); C) 10 mg of aphosphatidylcholine-derived compounds including[3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-hydroxypropyl]tetradecanoate,[3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-hydroxypropyl]pentadecanoate, or[3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-hydroxypropyl] hexadecanoate,structurally similar derivatives thereof, or thosephosphatidylcholine-derived compounds that are shown in FIG. 13 ; or D)placebo, respectively. Fecal samples for microbiome and lipid metaboliteprofiling are collected at days 7, 14, 21, and 28. Blood samples forimmunological and lipid metabolite profiling are collected at days 7 and28.

The data demonstrate that mice treated with bacteria capable ofproducing phosphatidylcholine-like compounds show significantly reducedinflammation. This is found for mice treated with both AM-ST7 and thebacterial consortium comprising AM-ST7 as well as FP-ST38, and LC-ST100.Fecal and blood sample analysis support these data by showingsignificantly reduced amounts of pro-inflammatory markers and increasedamounts of anti-inflammatory compounds. Particularly the amount ofproduced phosphatidylcholine and/or phosphatidylcholine-like compoundsmay correlate with a reduction in disease burden.

In sum, this study demonstrates that bacteria producingphosphatidylcholine and/or phosphatidylcholine-like compounds protectagainst inflammation.

Example 18 Microbial Consortia Producing Phosphatidylcholine-likeCompounds Protect Against Allergic Airway Inflammation

This example demonstrates that therapeutic microbial consortia of thepresent disclosure can alter the metabolism of certain compounds in asubject, resulting in increased therapeutic function of these consortia.

A pharmaceutical composition comprising a consortium comprising anAkkermansia sp. Faecalibacterium sp., and/or Lactobacillus sp. isadministered to animals suffering from an inflammatory disease inducedin a cockroach allergen (CRA) murine model. The mouse model was used asdescribed in EXAMPLE 6.

The animals receiving the therapeutic consortium show significantlyreduced inflammation compared to control animals. Analysis of tissuesamples (e.g., blood samples) indicate that animals treated with thetherapeutic consortium comprise higher amounts of anti-inflammatorycompounds and metabolites such as alpha-linolenic acid, and reducedamounts of pro-inflammatory compounds and metabolites such asarachidonic acid as a metabolite of alpha-linolenic acid.

This data indicates that, in addition to the production ofanti-inflammatory compounds, the therapeutic microbial consortia of thepresent disclosure can also alter the metabolism of certain compounds ina subject, in a way that reduces the ratio of anti-inflammatorycompounds or metabolites to pro-inflammatory compounds or metabolites ina subject, resulting in the treatment and/or prevention of a disease orcondition in a subject.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1.-72. (canceled)
 73. A pharmaceutical composition, wherein saidpharmaceutical composition comprises at most about 50% of Akkermansiamuciniphila DSM 33213 by weight of said pharmaceutical composition, andwherein said pharmaceutical composition is formulated as an oral dosage.74. The pharmaceutical composition of claim 73, wherein said oral dosageform comprises a liquid dosage form.
 75. The pharmaceutical compositionof claim 73, wherein said pharmaceutical composition is lyophilized orfrozen.
 76. The pharmaceutical composition of claim 75, wherein saidpharmaceutical composition is lyophilized.
 77. The pharmaceuticalcomposition of claim 75, wherein said pharmaceutical composition isfrozen.
 78. The pharmaceutical composition of claim 73, furthercomprising Lactobacillus sp. or Faecalibacterium sp.
 79. Thepharmaceutical composition of claim 78, further comprising saidLactobacillus sp. and said Faecalibacterium sp.
 80. The pharmaceuticalcomposition of claim 78, further comprising Lactobacillus crispatus orFaecalibacterium prausnitzii.
 81. The pharmaceutical composition ofclaim 78, further comprising Lactobacillus crispatus andFaecalibacterium prausnitzii.
 82. A method for modulating an immuneresponse in a subject, comprising administering to said subject acomposition comprising (1) Akkermansia sp. that produces a phospholipidor (2) said phospholipid, wherein in said phospholipid, an amine groupis attached to an oxygen atom of a phosphate group via an ethyl group,and wherein, when assayed from 7 days to 28 days subsequent to saidadministering: (1) a fecal sample of said subject has an increased levelof said phospholipid, relative to a level of said phospholipid in afecal sample of a subject not administered with said composition; or (2)a blood sample of said subject has an increased level of saidphospholipid, relative to a level of said phospholipid in a blood sampleof said subject not administered with said composition.
 83. The methodof claim 82, wherein, when assayed at 7 days, 14 days, 21 days, or 28days subsequent to said administering, (1) said fecal sample of saidsubject has said increased level of said phospholipid, relative to saidlevel of said phospholipid in said fecal sample of said subject notadministered with said composition; or (2) said blood sample of saidsubject has said increased level of said phospholipid, relative to saidlevel of said phospholipid in said blood sample of said subject notadministered with said composition.
 84. The method of claim 82, wherein,when assayed from 7 days to 28 days using mass spectroscopy subsequentto said administering: (1) said fecal sample of said subject has saidincreased level of said phospholipid, relative to said level of saidphospholipid in said fecal sample of said subject not administered withsaid composition; or (2) said blood sample of said subject has saidincreased level of said phospholipid, relative to said level of saidphospholipid in said blood sample of said subject not administered withsaid composition.
 85. The method of claim 82, wherein said compositionis administered to said subject as an oral dosage form.
 86. The methodof claim 82, wherein said subject is less than about 24 months old. 87.The method of claim 86, wherein said subject is a neonate.
 88. Themethod of claim 82, wherein said subject has a disease or a risk of saiddisease, wherein said disease comprises dysbiosis, inflammatory disease,autoimmune disorder, infection, cancer, or any combination thereof. 89.The method of claim 88, wherein said disease comprises said inflammatorydisease.
 90. The method of claim 89, wherein said inflammatory diseasecomprises an acne vulgaris, an Addison's disease, an allergic asthma, anallergy, an ankylosing spondylitis, an arthritis, an asthma, anatherosclerosis, an atopic dermatitis, an atopy, an autoimmune disease,an auto-immune thyroiditis, an autoinflammatory disease, a Behcet'sdisease, a bullous pemphigoid, a Celiac disease, a chronic persistentdiarrhea, a chronic prostatitis, a colitis, a collagenous colitis, aCrohn's disease, a diabetes mellitus type 1, a diverticulitis, anerythema nodosum, a glomerulonephritis, a Graves ophthalmopathy, aGuillain-Barre syndrome, a Hashimoto's encephalitis, a Hashimoto'sthyroiditis, a hypersensitivity, an ichthyosis, an immunoproliferativesmall intestinal disease, an inflammatory bowel disease, an interstitialcystitis, an intractable diarrhea of infancy, an irritable bowelsyndrome, a juvenile idiopathic arthritis, a juvenile onset diabetes, alymphocytic colitis, a multiple sclerosis, a myasthenia gravis, apediatric allergic asthma, a pelvic inflammatory disease, apostenteritis syndrome, a psoriasis, a psoriatic arthritis, a pyodermagangrenosum, a reperfusion injury, a rheumatoid arthritis, asarcoidosis, a sarcoidosis, a short bowel syndrome, a Sjogren'ssyndrome, a stagnant loop syndrome, a systemic lupus erythematosus(SLE), a transplant rejection, a traumatic brain injury, a Traveler'sdiarrhea, a tropical sprue, an ulcerative colitis, an uveitis, avasculitis, a Vitiligo, a Whipple's disease, or a Wolman disease. 91.The method of claim 82, wherein said composition is lyophilized orfrozen.
 92. The method of claim 82, wherein said composition furthercomprises Lactobacillus sp. or Faecalibacterium sp.